Method for preparing (αS,5S)-α-amino-3-chloro-2-isoxazoline-5-acetic acid (AT-125) and analogs thereof

ABSTRACT

Chemical process for preparing AT-125 and its analogs and intermediates used in the process.

CROSS REFERENCE TO RELATED CASES

This application is a continuation-in-part of application Ser. No.020,141, filed Mar. 13, 1979, which in turn is a division of Ser. No.905,963, filed May 15, 1978, both now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns a novel method for preparing (αS,5S)-α-amino-3-chloro-2-isoxazoline-5-acetic acid (AT-125) and novelanalogs thereof. The invention also includes novel intermediates formaking AT-125 and said analogs, novel compositions prepared from andmethods of using AT-125 and said analogs.

2. Description of the prior art

The compound AT-125 and the microbiological process for producing it areclaimed in U.S. Pat. Nos. 3,856,807 and 3,878,047, respectively. Thesepatents also disclose the antitumor and antimicrobial activity ofAT-125.

SUMMARY OF THE INVENTION

The novel compounds of this invention are selected from the groupconsisting of racemic mixtures and optically active isomers of compoundshaving the formula ##STR1## wherein R is selected from the groupconsisting of hydrogen, alkyl of from 1 to 8 carbon atoms, inclusive,halogenated alkyl of from 1 to 3 halogen atoms, and 1 to 5 carbon atoms,inclusive, and aralkyl of from 7 to 20 carbon atoms, inclusive;

X is selected from the group consisting of bromine, chlorine, fluorineand iodine, --OR₁, --SR₁, and --NR'R" wherein R₁ is selected from thegroup consisting of alkyl of from 1 to 12 carbon atoms, inclusive, arylof from 6 to 20 carbon atoms, inclusive; aralkyl of from 7 to 20 carbonatoms, inclusive; R' and R" are the same or different and are selectedfrom the group consisting of hydrogen and alkyl of from 1 to 8 carbonatoms; R₁₄ and R₁₅ are selected from the group consisting of hydrogen,##STR2## or when taken together with the nitrogen atom or the group##STR3## wherein R₆ is alkyl of from 1 to 8 carbon atoms, inclusive,halogenated alkyl of from 1 to 5 carbon atoms, inclusive, and 1 to 3halogen atoms, inclusive, aralkyl of from 7 to 20 carbon atoms,inclusive, and substituted aralkyl of from 7 to 20 carbon atoms,inclusive, R₇ is selected from the group consisting of alkyl of from 1to 12 carbon atoms, inclusive, aryl of from 6 to 20 carbon atoms,inclusive, aralkyl of from 7 to 20 carbon atoms, inclusive, andsubstituted aralkyl of from 7 to 20 carbon atoms, inclusive; and R₈ isselected from the group consisting of ##STR4## where R₉, R₁₀, R₁₁ andR₁₂ are selected from the group consisting of hydrogen and alkyl of from1 to 5 carbon atoms, inclusive, (b) ##STR5##

(c) orthointerphenylenes ##STR6## and (d) substitutedorthointerphenylene with the proviso that when R, R₁₄ and R₁₅ are allhydrogen, X cannot be chlorine, and the further proviso that when R₁₄and R₁₅ are both hydrogen and R₈ is orthointerphenylene, R can not behydrogen or alkyl of from 1 to 8 carbon atoms.

In the foregoing designation of variables,

"Lower-alkyl" means methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl and the isomeric forms thereof.

Halogenated alkyl of from 1 to 5 carbon atoms, inclusive, and 1 to 3carbon atoms, inclusive, means methyl, ethyl, propyl, butyl, pentyl andisomeric forms thereof substituted by 1 to 3 bromine, fluorine, chlorineor iodine atoms. "Aryl" means phenyl and phenyl containing 1 to 3substituents, the same or different and selected from the groupconsisting of halogen, alkoxy, alkyl and nitro. "Aralkyl" means benzyl,phenethyl, phenpropyl, phenbutyl, phenpentyl, diphenylmethyl, threediphenyloctyl and isomeric forms thereof and fluorenylmethyl.

"Substituted aralkyl" means aralkyl in which the phenyl ring or ringscontain 1 to 3 substituents, the same or different, and selected fromthe group consisting of halogen, alkoxy, alkyl and nitro. For example,p-methoxybenzyl, m-methoxybenzyl, p-nitrobenzyl, o-chlorobenzyl,4-propyl-2-methylbenzyl, 2-chloro-4-methylbenzyl, 3,4-diethoxybenzyl,3,4-diethoxybenzyl, 3,4,5-trichlorobenzyl and 3,4,5-trimethylbenzyl.

"Substituted orthointerphenlene" means lower-alkyl, loweralkoxy,halogen, nitro, and cyano substituted orthointerphenylene. There can becombinations of substituents such as 4-propyl-2-methyl-,2-chloro-4-methyl-3,4-diethoxy-3-cyano-4-ethoxy-phenyl and the like. Thesubstituted phenyl is limited to a total of 10 carbon atoms.

Halogen means bromine, chlorine, fluorine and iodine.

This invention also pertains to a chemical process for preparing AT-125and novel analogs thereof.

The process of this invention is advantageous in that it provides acomplete chemical synthesis of AT-125, which heretofore, had only beenprepared by a microbiological process. It involves preparing thecompounds dl-trans-3-amino-4-hydroxy-cyclopentene which is in turnconverted by a series of reactions to yield tricholomic acidderivatives, AT-125 and bromo, fluoro and iodo analogs thereof. Thesehalo compounds are then converted to other analogs.

Tricholomic acid, first isolated by Takemoto et al, see Yakugaku Zasshi,84, 1183 and 1230 (1964), is an amino acid having the structure ##STR7##

The compound is known to be flycidal and to have a taste that isappeallng to man.

In addition to the Takemoto et al. work, other methods for thepreparation of tricholomic acid have been described. They include[lwasaki et al., Chem., Pharm. Bull. 17(5) 866-872 (1969)], [lwasaki etal., Chem. Pharm. Bull 17(5) 873-878 (1969)], [Kamiya, T., Chem. Pharm.Bull. 17(5) 879-885 (1969)], [Kamiya, T., Chem. Pharm. Bull. 17(5)886-889 (1969)], [Kamiya, T., Chem. Pharm. Bull. 17(5) 890-894 (1969)],[Kamiya, T., and Chem. Pharm. Bull. 17(5) 895-900 (1969)].

AT-125, or (α5, 5S)-α-amino-3-chloro-α-isoxazoline-5-acetic acid, anantitumor and antimicrobial agent has the formula ##STR8##

The novel process which proceeds through tricholomic acid andderivatives thereof, can be represented schematically as follows:##STR9##

Another aspect of this invention is the use of novel intermediates inthe process set forth above to prepare the compounds of formula I. Theseintermediates include

(i) Compounds selected from the group consisting of racemic mixtures andoptically active isomers of compounds having the formula ##STR10##wherein R is selected from the group consisting of alkyl of from 1 to 12carbon atoms, inclusive, halogenated alkyl of from 1 to 5 carbon atoms,inclusive, and 1 to 3 halogen atoms, inclusive, aryl of from 6 to 20carbon atoms, inclusive, aralkyl of from 7 to 20 carbon atoms,inclusive, and substituted aralkyl of from 7 to 20 carbon atoms,inclusive; R₁₃ is selected from the group consisting of hydrogenalkoxycarbonyl, halogenated alkoxycarbonyl and aralkoxycarbonyl; R₁₄ andR₁₅ are selected from the group consisting of hydrogen, ##STR11## orwhen taken together with the nitrogen atom form the group ##STR12##wherein R₆ is alkyl of from 1 to 8 carbon atoms, inclusive, halogenatedalkyl of from 1 to 5 carbon atoms, inclusive, and 1 to 3 halogen atoms,inclusive, or alkyl of from 7 to 20 carbon atoms, inclusive, substitutedaralkyl of from 7 to 20 carbon atoms, inclusive, substituted aralkyl offrom 7 to 20 carbon atoms, inclusive, R₇ is selected from the groupconsisting of alkyl of from 1 to 8 carbon atoms, inclusive, aryl of from6 to 20 carbon atoms, inclusive, aryl of from 6 to 20 carbon atoms,inclusive, aralkyl of from 7 to 20 carbon atoms, inclusive, substitutedaralkyl of from 7 to 20 carbon atoms, inclusive, and R₈ is selected fromthe group consisting of ##STR13## where R₉, R₁₀, R₁₁ and R₁₂ areselected from the group consisting of hydrogen and alkyl of from 1 to 5carbon atoms, inclusive, ##STR14## orthointerphenylene and substitutedorthointerphenylene, the proviso that other than when R₁₄ and R₁₅ form aring with the nitrogen atom, one of R₁₄ and R₁₅ must always be hydrogen;when R₁₃, R₁₄, and R₁₅ are all hydrogen, R can not be hydrogen or alkylof from 1 to 8 carbon atoms; and that when R₁₃ is aralkoxycarbonyl orhalogenated alkoxy carbonyl, neither ##STR15## can be aralkoxycarbonylor halogenated alkoxycarbonyl, nor ##STR16## be aralkylcarbonyl orhalogenated alkylcarbonyl;

(ii) Compounds selected from the group consisting of racemic mixturesand optically active isomers of compounds having the formula ##STR17##wherein R₃ is selected from the group consisting of hydrogen and##STR18## where Ra is alkyl of 1 to 8 carbon atoms and halogenated alkylof 1 to 5 carbon atoms, inclusive, and 1 to 3 halogen atoms, inclusive,and aryl of from 6 to 20 carbon atoms, inclusive, and R₄ and R₅ are thesame or different and are selected from the group consisting ofhydrogen; ##STR19## wherein. R"₆ and R"₇ are selected from the groupconsisting of alkyl of from 1 to 8 carbon atoms, inclusive, halogenatedalkyl of from 1 to 5 carbon atoms, inclusive, and 1 to 3 halogen atoms,inclusive, aralkyl of from 7 to 20 carbon atoms, inclusive; substitutedaralkyl of from 7 to 20 carbon atoms, inclusive, and when taken togetherwith the nitrogen group ##STR20## wherein R₈ is selected from (a) thegroup consisting of ##STR21## where R₉, R₁₀, R₁₁ and R₁₂ are selectedfrom the group consisting of hydrogen and alkyl of from 1 to 5 carbonatoms, inclusive, (b) ##STR22## (c) orthointerphenylene and (d)substituted orthointerphenylenes.

(iii) Compounds selected from the group consisting of racemic mixturesand optically active isomers of compounds having the formula ##STR23##wherein Rc is selected from the group consisting of amino;di-alkylamino, ##STR24## wherein R"₈ and R"₇ are selected from the groupconsisting of alkyl of from 1 to 8 carbon atoms, inclusive, halogenatedalkyl of from 1 to 5 carbon atoms, inclusive, and 1 to 3 halogen atoms,inclusive, aralkyl of from 7 to 20 carbon atoms, inclusive, substitutedaralkyl of from 7 to 20 carbon atoms, inclusive, and the group ##STR25##wherein R₈ is selected from (a) the group consisting of ##STR26## whereR₉, R₁₀, R₁₁ and R₁₂ are selected from the group consisting of hydrogenand alkyl of from 1 to 5 carbon atoms, inclusive, (b) (c)orthointerphenylene and (d) substituted orthointerphenylene; R₄ and R₅are the same or different and are selected from the group consisting ofhydrogen, inclusive, or when taken together with the nitrogen atom formthe group ##STR27## wherein R"₆, R"₇ and R₈ are the same as above; withthe proviso that the groups R₃ and NR₄ R₅ are never simultaneously thesame.

The invention also pertains to compounds selected from the groupconsisting of racemic mixtures and optically active isomers of compoundshaving the formula ##STR28## wherein R₃ is the same as above.

DETAILED DESCRIPTION OF THE INVENTION

AT-125 and novel derivatives thereof can be prepared in accordance withthe process outlined above.

Step 1 is carried out in one of the three ways shown in the followingschematic diagram: ##STR29## In the preferred route shown as Step a,epoxy cyclopentene V, available as described, for example, by Crandallet al., J. Org. Chem., 33:423 (1968), is reacted with a solution ofammonia in protic solvents such as water, methanol, ethanol or otheralcohols or in non protic solvents such as diethyl ether, dimethylformamide, tetrahydrofuran, or dimethoxy ethane. The reaction in theaprotic solvent is most efficaciously run in the presence of a catalystsuch as dry, basic alumina as described for a similar reaction by Posnerand Rogers, J. Amer. Chem. Soc., 99:8214 (1978). The reaction in proticsolvents is generally run at -50° C. to +50° C. and at a concentrationof epoxide from 0.01M to 2M. The molar ratio of ammonia to epoxide isgenerally from 1:1 to 50:1 most preferably in the range of 15:1 to 20:1.In non protic solvents the reaction is generally run at -20° to 100°.The concentration of epoxide in these solvents is generally 0.01 to 2Mand the mole ratio of ammonia to epoxide is generally 1:1 to 20:1, mostpreferably 1:1 to 5:1. The amine IVb so produced is generally isolatedby evaporation of the excess ammonia and solvent and forming acrystalline salt of the crude residual amine. These salts may be formedusing a solution of an acid such as toluenesulfonic acid, hydrochloricacid, hydrobromic acid, hydroiodic acid or other acids in a solvent suchas water, methanol, ethanol, ether, 1,2-dimethoxy ethane, or p-dioxane.The salt is isolated by filtration and it crystallizes directly or byevaporation of the solvent followed by subsequent crystallization from asuitable solvent.

Alternatively, the crude amine IVb may be purified by adsorption on to acolumn of an acidic ion exchange resin such as Dowex 50W-X2(H+) orAmberlite 1R-120 (H⁺) followed by elution with a solvent such as water,methanol, ethanol, ether, tetrahydrofuran, 1,2-dimethoxy ethane orp-dioxane, containing a volative amine such as ammonia, methyl amine,diethyl amine or triethyl amine followed by evaporation of the volatileamine and solvent.

Similarly, the salts of hydroxy amine may be reconverted to the freeamine by passing a solution of the salt in a solvent such as water,methanol, ethanol, tetrahydrofuran or 1,2-dimethoxy ethane through abasic ion exchange resin such as Dowex 1-X8 (OH⁻) or Amberlite 1RA-400(OH⁻) and evaporating the eluate containing the amine.

The salts of amine IVb may also be reconverted to the free amine bytreating an aqueous solution of the salts with a slight molar excess ofa base such as sodium or potassium hydroxide saturating the resultantaqueous solution with a salt such as sodium chloride or sodium sulfateand subjecting the solution to constant solvent extraction with asolvent such as methylene chloride or chloroform. The amine IVb is thenisolated by evaporating the solvent.

In a second route to amine IVb, as shown in the schematic diagram stepb, the epoxide V is reacted with a mixture of hydrazoic acid and1,1,3,3-tetramethyl guanidine in a solvent such as methylene chloride.

There are many references to the opening of epoxides with azide ion. SeeJ. Org. Chem. 37, 1268 (1972); J. Am. Chem. Soc., 93, 1813 (1971); J.Org. Chem. 32, 1452 (1967); J. Med. Chem. 15, 175 (1972) and J. Am.Chem. Soc. 94, 7098 (1972). However, the high reactivity of the acyclicepoxide is unexpected. The use of 1,1,3,3-tetramethyl guanidine tosolubilize azide ion in organic solvent has been described by Papa, J.Org. Chem. 31, 1426 (1966). However, the specific opening of epoxides tohydroxy azides with the reactants HN₃ and 1,1,3,3-tetramethyl guanidinein methylene chloride has not to applicants knowledge been reportedbefore.

The molar ratios of hydrazoic acid and guanidine to epoxide should befrom 1:1 to 5:1 and from 1:100 to 1:1 respectively. The reaction can berun at temperatures between about -50° to 100 at concentrations from0.01 to 2 M. Under these conditions the dl-3,4-epoxycyclopentene isextremely reactive. Solvents other than methylene chloride that can beused include tetrahydrofuran, dimethylformamide p-dioxane and glyme. Theazide VI can be removed from the reaction mixture by conventionalseparation techniques, i.e. filtration, extraction, chromatography andcombinations thereof.

An alternative procedure for step b may be represented schematically asfollows ##STR30## In this reaction dl-3,4-epoxycyclopentene is reactedwith a mixture of sodium azide and a mild acid catalyst such as boricacid suspended in a solvent such as dimethylformamide, dimethylsulfoxideor acetonitrile. About 1 to 5 mole of sodium azide per mole of epoxideand 1 to 5 mole of boric acid per mole of epoxide is used. The reactionmay be conducted between the temperatures of 0° and 125° for a period ofabout 1 hour to 10 days. The desired productDL-trans-3-azido-4-hydroxycyclopentene VI is recovered from the reactionmixture by conventional methods such as extraction and chromatographyand combinations thereof.

On prolonged standing at room temperature and more rapidly on heatingthe azides VI and VIa each rearranges partially to the other azide. Thetwo isomers VI and VIa may be separated chromatographically.

Step c involves the reduction of thedl-trans-3-azido-4-hydroxycyclopentene VI todl-trans-3-amino-4-hydroxycyclopentene IVb. This type of reaction iswell known in the art. However superior results are more likely to beobtained in polar solvents which dissolves the highly polardl-trans-3-amino-4-hydroxycyclopentene. Suitable solvents includetetrahydrofuran, p-dioxane and 1,2-dimethoxyethane. The reaction can beconducted at temperatures of between -78° and 50° and concentration of0.01 to 2 M. The amine IVb can be obtained from the reaction mixture bythe methods described above for step a.

In the third route to IVb, step d, epoxide V is reacted with phthalimidein the presence of a catalyst such as potassium phthalimide or atertiary amine in a solvent such as dimethylformamide,dimethylsulfoxide, tetrahydrofuran or acetonitrile. The molar ratios ofphthalimide and catalyst to epoxide may be respectively, 1:1 to 10:1 and1:10 to 1:1. The reaction may be run at 0° to 125° and for 1 hour to 20days, the higher temperatures corresponding to shorter reaction times.The hydroxy phthalimide IVd can be obtained from the reaction mixture byconvention separation techniques, i.e. extraction chromatography andcombinations thereof.

In step e the phthalimide IVd is converted to the hydroxyamine IVb bytreatment with hydrazine or hydroxylamine as is known in the art.Suitable solvents for this reaction include alcohols such as methanoland ethanol, tetrahydrofuran, dioxane, dimethylformamide, andacetonitrile. The molar ratio of phthalimide IVd to hydrazine orhydroxylamine is generally 1:1 to 1:10 and the reaction may be run at-20° C. to 100° C., most preferably at or near room temperature. Theamino alcohol IVb may be isolated as obtained for step a above.

Step 2 involves reacting dl-trans-3-amino-4-hydroxycyclopentene IVb withtrichloroethylchloroformate. In this step the amino group is blocked bythe trichloroethoxycarbonyl group. The reaction is conducted by methodswell known in the art for converting amines to urethanes. The reactionis conducted using a molar ratio of trichloroethylchloroformate to IVbof about 1:1 to 10:1 and at a temperature of about -20° to 50° for aperiod of about 20 minutes to 24 hours. The reaction is generally run ina solvent such as water, methanol, methylene chloride, tetrahydrofuranas p-dioxane containing a dissolved or suspended base such as sodium orpotassium carbonate or a tertiary amine to capture the liberated acid.The reaction may also be run in a solvent which acts as its own basesuch as pyridine. The preferred solvent is water containing sodiumcarbonate. The urethane IVa can be recovered from the reaction mixtureby conventional methods such as crystallization, filtration,distillation, extraction, chromatography and combinations thereof.

Other halo substituted alkyl halo formates can be used to block theamino group, i.e. iodoethylchloroformate. Also, the amino group can beblocked by an aralkoxycarbonyl group or an alkyloxycarbonyl group forexample benzyloxycarbonyl and t-butyloxy carbonyl respectively. Methodsfor using these groups to block amines are well known in the art. Forexample background information on the preparation and removal ofphthalimide, p-nitrobenzyl esters of carbobenzyloxy andcarbo-tert-butyloxy derivatives of amino acids is described by R. A.Boissonas Chapter, "Selectively Removable Amino Protective Group used inthe Synthesis of Peptides." In: Advances in Organic Chemistry, 3:159-190(1963). Information on the use of the t-butyloxycarbonyl group to blockamine is also described in ALDRICH Technical Information Bulletinentitled BOC-ON (September, 1976). Information on the use oftrichloroethoxycarbonyl to block amines is disclosed by Windholz et al.,Tetrahedron Letters, 2555 (1967). However the particular sequence usedby the instant inventors have not to their knowledge been used before.

The urethanes IVa can also be prepared from salts ofdl-trans-3-amino-4-hydroxycyclopentene formed by reacting the amine withprotonic acids. The reaction is conducted by dissolving the salt inwater and then treating the solution with sodium carbonate andtrichloroformate or another blocking agent of the type described in step2 above. The urethanes IVa again can be recovered from the mixture byconventional means.

Step 3 is conducted by reacting the urethane IVa with a slight molarexcess of N-hydroxyphthalimide, diethyl azodicarboxylate andtriphenylphosphine in a solvent. The reaction is conducted at atemperature of about -50° to 50°. Suitable solvents includetetrahydrofuran, ether, 1,2-dimethoxy ethane and p-dioxane. Thepreferred solvent is tetrahydrofuran. It should be noted that aninversion occurs during step 3 and that the urethane III_(e) obtained isa cis isomer.

With slight modifications, other compounds, formed from the amine andother halo substituted alkyl halo formates, can be utilized in place ofthe dl-trans-3-amino-4-hydroxycyclopentene-N-trichloroethyl urethane instep 3.

Also compounds wherein the amino group is blocked by an aralkoxycarbonylgroup can be used in step 3. For example, ##STR31## The reaction andrecovery procedure is essentially the same as in 3 above.

In step 4 the phthalimide compound IIIe is converted to the hydroxylamine III_(d) by reacting it with a slight molar excess of hydrazinehydrate. This reaction is conducted in the presence of a solvent at atemperature of between -20°and 100° for a period of about 1 hour to 2days. Solvents that can be used include tetrahydrofuran, ethanol,methanol, water, p-dioxane, and dimethylformamide. The hydroxyl amineIIId can be recovered from the reaction mixture by conventional meanssuch as extraction, crystallization chromatography and combinationsthereof.

In step 5 the hydroxylamine group of compound IIId is protected byreacting it with an alkoxychloroformate, haloalkoxychloroformate or anaralkoxyhaloformate such as benzyloxychloroformate to yield thecarbamate IIIc. The reaction is conducted by using a molar ratio ofaralkoxyhaloformate to IIIg of about 1:1 to 10:1 in a solvent such astetrahydrofuran, 1,2-dimethoxy ethane, p-dioxane, acetonitrile ordimethylformamide containing a tertiary amine base such as triethylamine or in a solvent such as pyridine which acts as its own base. Thepreferred solvent is pyridine. The reaction is generally run at atemperature of -20° to 50° C.

Conventional means such as crystallization, extraction, chromatographyand combinations thereof can be used to recover the product from thereaction mixture.

In step 6, the amino group of the compound IIIc is deprotected byreacting it with zinc to yield the amino carbamate IIIb. This reactionmay be conducted and worked up by several methods.

The preferred method involves conducting the reaction in the presence ofmethane sulfonic acid and methanol. The reaction is conducted at atemperature of between 0° C. and 50° C. for a period of about 30 minutesto 5 hours, using a molar ratio of zinc acid to IIIc of about 2:1 to50:1.

In reaction 7 the amino carbamate IIIb is converted to its phthalimideIIIa by reacting it with 2-methoxy carbonylbenzoyl chloride andtriethylamine in the presence of a solvent to yield phthalimide productIIIa. The reaction is conducted at a temperature of between -20° and 50°for a period of about 10 minutes to 5 hours using about 1:1 to 10:1molar ratio of the benzoylchloride and 1:1 to 20:1 molar ratio oftriethylamine to IIIc. Suitable solvents include tetrahydrofuran, ether,1,2-dimethoxyethane, p-dioxane, dimethylformamide, and methylenechloride. The product IIIa is recovered from the reaction mixture byconventional means such as extraction, crystallization, chromatographyand combinations thereof. The preferred method of recovery is extractionfollowed by chromatography or crystallization.

The starting material 2-methoxy carbonylbenzoyl chloride can be preparedby a method disclosed by Hoogwater et al., Recueil, 92, 819 (1973).

In step 8 the alkene phthalimide IIIa is reacted with a rutheniumcompound such as ruthenium trichloride in the presence of an oxidizingagent such as potassium or sodium iodate and a solvent to yield thebenzyloxycarbonyl derivative of tricholomic acid IIb. This reaction isconducted utilizing molar ratios of ruthenium chloride and sodium iodateto IIIa of about 1:1000 to 1:10 and 4:1 to 10:1 respectively, and at atemperature of about 0° to 50° for a period of about 20 minutes to 24hours. Suitable solvents include water with acetone, methyl acetate,nitromethane, or t-butyl alcohol. The preferred solvent isacetone-water. The product IIb can be recovered from the reactionmixture by conventional means.

A product having the formula ##STR32## is also formed during reaction 8.

In step 9 the compound IIb is prepared by the deprotection of compoundIIc. The particular conditions of the deprotection depend upon theparticular oxycarbonyl groups, group (R₁₃) that is attached to nitrogenatom of the tricholomic acid ring. When that group is benzyloxy oraralkoxycarbonyl the deprotection can be conducted by dissolving thecompound in a solvent and treating the solution with hydrogen in thepresence of a conventional catalyst at a temperature of between 0° and50° C. and atmospheric pressure for a period of about 20 minutes to 2hours. Suitable solvents include ethyl acetate, ethanol, toluene andtetrahydrofuran. Suitable catalyst include palladium block, 5% palladiumon carbon and palladium on barium carbonate. The product IIa can berecovered by conventional means such as extraction, crystallization,chromatography and combinations thereof.

Alternatively, deprotection of compounds wherein R₁₃ is alkoxycarbonylor aryloxycarbonyl can be conducted in the presence of an acid insolvents such as nitromethane and methylene chloride.

When R₁₃ is haloalkoxycarbonyl the deprotection is preferably conductedin the presence of zinc.

In step 10 the tricholomic acid derivative (IIb) is converted to esterIIa by methods for esterification well known in the art. For example theacid may be treated with diazo alkane or arylated diazomethane, forexample diphenyl diazomethane or by treating IIa with a hinderedtertiary amine such as N,N-diisopropyl-N-ethylamine followed by a benzylhalide such as diphenyl methyl chloride or p-methoxy benzyl bromide in asolvent such as tetrahydrofuran, ethyl acetate, acetonitrile ordimethylformamide.

In step 11 the tricholomic acid ester IIa is subjected to chlorinationto yield the phthalimide-isoxozole acetic acid ester Ic. The preferredmethod of chlorition involves reacting II with hexamethylphosphoroustriamide dichloride in the presence of a solvent. This reaction can beconducted at a temperature of between 25 and 60 for a period of about24-72 hours. The molar ratios of hexamethylphosphorous triamidedichloride to IIa can be from 1 to 3. Wolkoff, CAN. J. CHEM. VOL. 53, p.1333 (1975) discloses a process for converting benzoyl hydrogens intotheir corresponding hydrazonyl halides. However, attempts to converttrichalomic acid, which contains an oxyamide group to its correspondinghydrazonyl halide compounds using this method were unsuccessful.

In step 12 the ester Ic is converted to Ib by either deesterification ordeprotection of the amine. The preferred method is to performdeesterification first.

Deesterification of Ib involves reacting it with a hydrohalide gas inthe presence of a solvent. The reaction is conducted at a temperature ofabout 5° to 30° for a period of about 1 through 4 hours. Suitablesolvents include nitromethane, acetic acid and methylene chloride. Thepreferred solvent is nitromethane. The halogen moiety X₁ is dictated bythe particular hydrogen halide used. For example, the use of hydrogenbromide and hydrogen chloride yields compounds of Formula Ib wherein X₁is bromo and chloro, respectively.

The deprotection of the phthalimide amino acid produced by thedeesterification of the ester Ic is accomplished by reacting thedeesterification product with hydrazine hydrate in water or alcohol. Theproduct (α5,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid orAT-125 (when X₁ is chloro) is recovered from the reaction mixture byconventional means such as extraction, crystallization, chromatographyand combinations thereof.

Compounds of Formula Ia, Ia₂ and Ia₃ are prepared from compounds offormula Ia or IIa using conventional techniques for replacing halogenatoms with other moieties. For example: ##STR33## wherein R₁, R', R" andX₁ are the the same as defined above.

The reaction of Ia in methanol with one equivalent of sodium methoxideunder an inert atmosphere at room temperature for 1-40 hours affords Iawherein R₁ =methyl. Alternatively, a suspension of Ia in an inertsolvent such as dimethylformamide treated with an equivalent of analkali metal salt of an alkyl or aryl mercaptan or primary or secondaryaryl/aryl amine can be employed to afford analogous products Ia₂ andIa₃, respectively.

The treatment of IIa with diazomethane in an ether solvent with acatalytic amount of boron trifluoride etherate gives Ia₁ with R₁=methyl.

The process of this invention requires the selective removal of variousprotecting groups in the presence of different protecting groups. Ingeneral, ease of removal of one group in the presence of anothergenerally depends upon the particular reagents used. In this respect theprotecting groups can be ranked in the order of least difficult toremove to most difficult to remove as shown in Table I.

    __________________________________________________________________________    Acid (H.sup.+) Hydrogen (H.sub.2)                                                                        Zn (metal)                                                                              Hydrazine                                __________________________________________________________________________      Substituted aralkoxy- carbonyl (i.e., p-methoxy- benzyloxy                                 Substituted aralkoxy- carbonyl                                                            Halogenated alkoxycarbonyl                                                               ##STR34##                                 Akloxycarbonyl (i.e.,                                                                      Aralkoxycarbonyl                                                                          Substituted*                                         t-butyloxycarbonyl)      aralkoxycarbonyl                                     Aralkoxycarbonyl (i.e.,                                                                    Alkoxycarbonyl*                                                                           alkoxycarbonyl*                                      benzyloxycarbonyl)                                                            Halogenated-alkoxycar- bonyl(i.e., trichloro- ethoxy carbonyl)*                            Halogenated carbonyl*                                                                      ##STR35##                                            ##STR36##                                                                                  ##STR37##                                                     __________________________________________________________________________     *Generally cannot be removed without destroying total molecule.          

Each step of the above process can either be conducted on racemicmixtures of the various reactants or a resolution can be conducted atany stage along the process and the remaining steps conducted uponoptically active reactants.

The preferred method is to resolve theDL-trans-amino-4-hydroxy-cyclopentene IVb or a protonic acid saltthereof and then conduct the remainder of the process on the opticallyactive isomers of the reactants.

Resolution of the racemic mixture can be accomplished, withmodifications obvious to those skilled in the art, utilizingconventional methods of resolution. For example, the compound IVb may beresolved by forming salts with optically active acids such as D or Ltartaric acid, the L-(+)-acid giving the antipode which leads to AT-125of the natural configuration. The respective salts are obtainedoptically pure by recrystallization several times from a solvent such asethanol. A particularly effective method of obtaining the optically pureisomer of IVb leading to natural AT-125 is to treat the racemic IVb in asolvent such as methanol first with deoxycholic acid which crystallizesout predominantly the antipode leading to enantiomeric AT-125. Themother liquors from this crystallization are then reconverted to thefree amine by any of the methods described for step a above. Thisrecovered amine now substantially enriched in the desired antipode isthen brought to optical purity by formation and recrystallization of theL-tartaric acid salt.

Since the compounds I of this invention are amphoteric compounds, theyform salts with acids, alkali metals (including ammonia), alkaline earthmetals (including magnesium and aluminum), and amines. Metal salts canbe prepared by dissolving them in water, and adding a dilute metal baseuntil the pH of the solution is 7 to 8. Metal salts include the sodium,potassium and calcium salts. Amine salts, including those with organicbases such as primary, secondary, and tertiary, mono-, di-, andpolyamines can also be formed using the above-described or othercommonly employed procedures. Further, ammonium salts can be made, bywellknown procedures. Other salts are obtained with therapeuticallyeffective bases which impart additional therapeutic effects thereto.Such bases are, for example, the purine bases such as theophyllin,theobromin, caffein, or derivatives of such purine bases; antihistaminicbases which are capable of forming salts with weak acids; pyridinecompounds such as nicotinic acid amide, isonicotinic acid hydrazide andthe like; phenylalkylamines such as adrenalin, ephedrin, and the like;choline, and others.

Acid salts can be made by neutralizing compounds of formula I with theappropriate acid to below about pH 7.0, and advantageously to about pH 2to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric,phosphoric, sulfamic, hydrobromic, and the like. Acid and base salts ofthe compounds can be used for the same biological purposes as the parentcompound.

The compounds of formulas I and VIId inhibit the growth ofmicroorganisms in various environments. For example, AT-125 is activeagainst Escherichia coli and can be used to reduce, arrest, anderadicate slime production in papermill systems caused by itsantibacterial action against this microorganism. AT-125 also can be usedto prolong the life of cultures of Trichomonas foetus, Trichomonashominis, and Trichomonas vaginalis by freeing them of Escherichia colicontamination. Further, AT-125 can be used as the antifungal agent inthe shoe uppers as disclosed in U.S. Pat. No. 3,130,505. Still, further,since AT-125 is active against Bacillus subtilis it can be used tominimize or prevent odor in fish or fish crates caused by this organism,or AT-125 can be used to swab laboratory benches and equipment in amycological laboratory.

The compounds of formula I are also effective for treating bacterialinfections and tumors in mammals, including humans.

The compositions of the present invention are presented foradministration to humans and animals in unit dosage forms, such astablets, capsules, pills, powders, granules, sterile parenteralsolutions or suspensions, eye drops, oral solutions or suspensions, andwater-in-oil emulsions containing suitable quantities of the compound ofFormula I.

For oral administration, either solid or fluid unit dosage forms can beprepared. For preparing solid compositions such as tablets, the compoundof FIG. 1 is mixed with conventional ingredients such as talc, magnesiumstearate, dicalcium phosphate, magnesium aluminum silicate, calciumsulfate, starch, lactose, acacia, methylcellulose, and functionallysimilar materials as pharmaceutical diluents or carriers. Capsules areprepared by mixing the compound with an inert pharmaceutical diluent andfilling the mixture into a hard gelatin capsule of appropriate size.Soft gelatin capsules are prepared by machine encapsulation of a slurryof the compound with an acceptable vegetable oil, light liquidpetrolatum or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs,and suspensions can be prepared. The watersoluble forms can be dissolvedin an aqueous vehicle together with sugar, aromatic flavoring agents andpreservatives to form a syrup. An elixir is prepared by using ahydroalcoholic (ethanol) vehicle with suitable sweeteners such as sugarand saccharin, together with an aromatic flavoring agent.

Suspensions can be prepared with an aqueous vehicle with the aid of asuspending agent such as acacia, tragacanth, methylcellulose and thelike.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, water being preferred. Thecompound, depending on the vehicle and concentration used, can be eithersuspended or dissolved in the vehicle. In preparing sultions thecompound can be dissolved in water for injection and filter sterilizedbefore filling into a suitable vial or ampoule and sealing.Advantageously, adjuvants such as a local anesthetic, preservative andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection issupplied to reconstitute the liquid prior to use. Parenteral suspensionscan be prepared in substantially the same manner except that thecompound is suspended in the vehicle instead of being dissolved andsterilization cannot be accomplished by filtration. The compound can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

Additionally, a rectal suppository can be employed to deliver the activecompound. This dosage form is of particular interest where the mammalcannot be treated conveniently by means of other dosage forms, such asorally or by insufflation, as in the case of young children ordebilitated persons. The active compound can be incorporated into any ofthe known suppository bases by methods known in the art. Examples ofsuch bases include cocoa butter, polyethylene glycols (Carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.These rectal suppositories can weigh from about 1 to 2.5 Gm.

The term "unit dosage form", as used in the specification, refers tophysically discrete units suitable as unitary dosages for human subjectsand animals, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular effect to beachieved and (b) the limitations inherent in the art of compounding suchan active material for use in humans and animals, as disclosed in detailin this specification, these being features of the present invention.Examples of suitable unit dosage forms in accord with this invention aretablets, capsules, pills, suppositories, powder packets, wafers,granules, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampoules,vials, aerosols with metered discharges, segregated multiples of any ofthe foregoing, and other forms as herein described.

An effective quantity of the compound is employed in treatment. Thedosage of the compound for treatment depends on many factors that arewell known to those skilled in the art. They include for example, theroute of administration and the potency of the particular compound. Adosage schedule for humans of from about 2 to about 200 mg. of compoundin a single dose, administered parenterally or in the compositions ofthis invention, are effective for treating tumors and bacterialinfections. More specifically, the singe dose is from about 5 mg. toabout 50 mg. of compound. The oral and rectal dose is from about 5 mg.to about 500 mg. in a single dose. More specifically, the single dose isfrom about 10 mg. to about 100 mg. of compound.

The following described preparations of AT-125 and analogs thereof areindicative of the scope of this invention and are not to be construed aslimitative. Those skilled in the art will promptly recognize variationsfrom the procedure both to the AT-125 and analog precursors as well asreaction conditions and techniques.

PREPARATION OF RACEMIC MIXTURES

Preparation 1 dl-3,4-Epoxycyclopentene ##STR38##

A 45 g. (0.68M) quantity of cyclopentadiene is dissolved in 750 ml. ofmethylene chloride to which is added 290 g. of anhydrous sodiumcarbonate. The vigorously stirred mixture is treated dropwise over 40min. with 110 ml. of 6.3 M peracetic acid (to which 1 g. of sodiumacetate is added to neutralize possible traces sulfuric acid whileholding the temperature below 20° in an ice bath. The reaction is thenstirred an additional 3 hours at 20°-25°. The reaction is then filteredand 1-2 g. of sodium carbonate is added to the filtrate. The filtrate isdistilled starting at 35 mm of pressure and ending at 20 mm with adistilling temperature ˜30° (pot temperature never above 40°-45°) toyield 21.8 g. of dl-3,4-epoxycyclopentene.

NMR (CDCl₃, δ): 2.1-2.7 (m, 2H), 3.6-4.1 (m, 2H), 5.7-6.3 (m, 2H).

Preparation 2 dl-Trans-azido-4-hydroxycyclopentene ##STR39##

A 90 g. (1.38 M) quantity of sodium azide was added to 90 ml. of H₂ Oand 300 ml. of methylene chloride. The mixture is vigorously stirred,cooled to <10° and treated with 19 ml. of sulfuric acid. After 30 min.the methylene chloride layer is decanted and dried over sodium sulfate.The solution is then treated with 8 g. (0.07 M) of1,1,3,3-tetramethylguamidine dissolved in 30 ml. of methylene chloride.The resultant solution is treated with the crude methylene chloridesolution of 3,4-epoxycyclopentene prepared from 0.68 M ofcyclopendadiene. After two and one half hours the solvent is evaporatedin vacuo to yield DL-trans-azido-4-hydroxycyclopentene.

Preparation 2a dl-trans-azido-4-hydroxycyclopentene anddl-trans-azido-2-hydroxycyclopentene ##STR40##

In an alternate procedure the crude methylene chloride solution ofDL-3,4-epoxycyclopentene prepared from 36.6 mm of cyclopentadiene isconcentrated under ˜50 mm and <20°. The residue is added to a mixture of8 g. (123 mM) of sodium azide and 8 g. (129 mM) of Boric Acid suspendedin 200 ml. of dry dimethylformamide. The mixture is stirred at 25° for17 hours. The reaction is then partitioned between water and ethylacetate. The ethyl acetate layer is washed with water, dried overmagnesium sulfate and evaporated in vacuum at 25°. The crude residue iscombined with that from a previous 10 mM run and chromatographed over 2linearly connected E. Merck size B columns of silica gel 60. The columnsare eluted with (40-60) ethyl acetate-Skellysolve B and 30 ml. fractionswere collected. Fractions 10-13 are collected and evaporated to yieldleft 2.64 g of dl-trans-azido-4-hydroxycyclopentene as a pale yellowoil. The overall yield from cyclopentadiene is 45%.

On standing dl-trans-azido-4-hydroxycyclopentene rearranges partially todl-trans-azido-2-hydroxycyclopentene.

The two isomers are separated by chromatography over 2 linearlyconnected E. Merck size B columns of silica gel 60. The columns areeluted with (40-60) ethyl acetate-Skellysolve B and 30 ml. fractions arecollected.

dl-Trans-3-amino-4-hydroxy-cyclopentene

NMR (CDCl₃, δ): 1.95-3.05 (m, CH₂), 3.5 (OH), 4.0-4.5 (m, CHOH, CHN₃),5.6-6.2 (m, CH=CH).

IR (film, cm⁻¹): 3300 (OH), 2090 (azide).

dl-Trans-3-amino-2-hydroxy-cyclopentene

NMR (CDCl₃, δ): 1.9-2.2 (m, CH₂), 3.85-4.2 (OH), 4.25-4.7 and 4.7-5.1(m, CHOH, CHN₃), 5.75-6.2 (m, CH=CH).

Ir (film, cm⁻¹): 3300 (OH), 2090 (azide).

TLC (silica gel 60): Rf of isomer IVc is 0.60 and the Rf of isomer VI is0.39 in (40-60) ethyl acetate-Skellysolve B.

Preparation 3 dl-trans-3-amino-4-hydroxy-cyclopentene ##STR41##

(a) A 2.2 g. (57.9 mM) quantity of lithium aluminum-hydride is added to90 ml. of ether and the stirred suspension cooled in an ice bath. To theabove suspension is added 3.62 g. of pureDL-trans-azido-4-hydroxycyclopentene dissolved in 5 ml. of ether over 15min. (exothermic with vigorous gas evolution). After stirring 2 hours,the reaction is cautiously treated with 2.2 ml. of water, 2.2 ml. of 15%sodium hydroxide and 2.2 ml. of water (initially, very exothermic withmuch gas evolution). After stirring an additional 15 min. the solids areremoved by filtration. The filtrate is evaporated in vacuo leaving 1.96g. of dl-trans-3-amino-4-hydroxycyclopentene, 5 which solidified to alow melting solid on standing. This material is extremely water soluble.

NMR (CDCl₃, δ): 1.8-2.9 (m, CH₂), 3.2 (OH, NH₂), 3.5-4.2 (m, CHOH,CHNH₂), 5.4-5.9 (m, CH═CH).

TLC (silica gel 60): Rf=0.24 in (1-20-80) conc. NH₄ OH-MeOH-CH₂ Cl₂. (b)The total crude dl-trans-azido-4-hydroxycyclopentene from a 0.68 M scale1,1,3,3-tetramethylguanidine assisted azide formation (Preparation 2a)is dissolved in 100 ml. of tetrahydrofuran and the solution added over30 minutes to a suspension of 25.8 g (0.68 M) of lithium aluminumhydride in 1 l. of tetrahydrofuran. The temperature of the reaction ismaintained below 5° with a brine-ice bath during the addition. Thereaction is then allowed to warm to 25° C. and stirred for 18 hours. Thereaction is then recooled to 0° and treated successively with 25 ml. ofwater (exothermic and vigorous gas evolution), 25 ml. of 15% sodiumhydroxide, and 25 ml. of water. After stirring for an additional onehalf hour, the mixture is filtered. The solids are washed withtetrahydrofuran. The combined filtrate and washings are evaporated invacuo leaving 47.8 g. of crude dl-trans-3-amino-4-hydroxycyclopentene.

Preparation 4 dl-Trans-3-amino-4-hydroxycyclopentene toluenesulfonatesalt ##STR42##

A 22.17 g. quantity of crude dl-trans-3-amino-4-hydroxy-cyclopentenedissolved in ˜200 ml. of tetrahydrofuran and the solution treated with asaturated tetrahydrofuran solution containing 40 g. (0.21 M) ofp-toluenesulfonic acid monohydrate. Crystals form rapidly. They areremoved by filtration after a few minutes giving 14.5 g. ofdl-trans-3-amino-4-hydroxycyclopentene toluenesulfonate salt m.p.180°-182°. The mother liquors are concentrated and cooled giving another18.9 g. of dl-trans-3-amino-4-hydroxycyclopentene toluenesulfonate ascrystals m.p. 177°-181° for a total yield of 33.4 g.

Preparation 5 Preparation of dl-3-Amino-4-hydroxycyclopentene from itstoluenesulfate salt. ##STR43##

A 150 ml quantity of amberlite IRA-400 resin in its chloride form iswashed on a fritted glass funnel successively with 6×150 ml of N sodiumhydroxide, 3×150 ml of water and 3×150 ml of methanol. The beads arethen slurry packed in a chromatography column containing methanol. A10.95 g (40.4 mM) quantity of dl-trans-3-amino-4-hydroxycyclopentenetoluenesulfonate dissolved in a minimum amount of methanol is then addedto the top of the column and the column eluted with 750 ml of methanol.Evaporation of the total eluate gives 4.2 g. of an oil which solidifieson standing overnight under vacuum to givedl-3-amino-4-hydroxycyclopentene, m.p. 47°-50° C.

NMR (CDCl₃, δ): 1.8-2.9 (m, CH₂), 3.2 (OH, NH₂), 3.5-4.2 (m, CHOH,CHNH₂), 5.4-5.9 (m, CH═CH).

TLC (silica gel 60); Rf - 0.24 in (1-20-80) conc. NH₄ OH-MeOH-CH₂ Cl₂.Detected by KMnO₄ spray reagent.

Preparation 5a dl-Trans-3-amino-4-hydroxycyclopentene (IVb) and itsp-toluenesulfonate salt ##STR44##

A 40 ml. quantity of dry methanol is cooled in an ice bath and ammoniagas bubbled to produce a saturated solution. To this ammonia solution isadded 500 mg. of cyclopentene epoxide. The reaction is held at 0° for 24hours and at 25° for 64 hours. The reaction is then concentrated invacuo and the residue treated with 5 to 10 ml of tetrahydrofuran. Theinsoluble material is removed by centrifugation leaving a solution ofhydroxy amine IVb. This is treated in a saturated solution ofp-toluenesulfonic acid in tetrahydrofuran until the solution containingthe amine becomes acidic. The voluminous crystalline precipitate iscollected by filtration and dried. There is obtained 680 mg. ofdl-trans-3-amino-4-hydroxycyclopentene toluenesulfonate salt.

Preparation 5b dl-Trans-3-amino-4-hydroxycyclopentene IVb and itsp-toluenesulfonate salt ##STR45##

A 1.97 g. (24 mM) quantity of distilled epoxycyclopentene is added to anice cold solution of 1.97 g. (39.4 mM) ammonium chloride in 20 ml. ofconc. ammonium hydroxide. After stirring one half hour the reaction iswarmed to 25° and stirred an additional 18 hours. The reaction is thenextracted with ether to remove more polar impurities. The aqueous layeris saturated with sodium chloride subjected to continuous extractionwith methylene chloride. The methylene chloride is dried over sodiumsulfate and concentrated in vacuo leaving 1.15 g. ofdl-trans-3-amino-4-hydroxycyclopentene which is treated with 1.54 g.(8.1 mM) of toluene sulfonic acid dissolved in tetrahydrofuran to yield1.47 g. of dl-trans-3-amino-4-hydroxycyclopentene toluenesulfonate.

Utilizing a procedure similar to Preparation 5a but substituting theappropriate protonic acid for toluenesulfonic acid there is obtainedracemic mixtures of

di-p-tolyl-L-tartaric acid salt, m.p. 185°-190° (dec).

di-benzoyl-L-tartaric acid salt, m.p. 181°-183° (dec).

1-mandelic acid salt, m.p. 116°-117°.

2-pyrrolidone-5-carboxylic acid salt, m.p. 164°-172°.

Preparation 6 dl-Trans-4-hydroxy-3-phthalimidocyclopentene ##STR46##

A quantity of dl-trans-4-hydroxy-3-amino-cyclopentene is dissolved in450 ml. of tetrahydrofuran and 87 ml. (0.60 M) of triethylamine. Thesolution is cooled to 0° and treated dropwise with 59.6 g. (0.3 M) ofo-methoxy-carbonylbenzoylchloride over 50 min. The reaction is thenallowed to warm to 25°. After 66 hours the reaction is partitionedbetween ethyl acetate and water. The ethyl acetate layer is dried overmagnesium sulfate and distilled in vacuo to yield 62.4 g. of residualoil. This is chromatographed over 3 kg. of silica gel 60 eluted with 6l. of (10-90), 2.5 l. of (15-85), and 11 l. of (20-80) acetonemethylenechloride. Two hundred fifty ml. fractions are collected. The product isfound in fractions 38-61. These fractions crystallized on standing toyield 25.9 g. of dl-trans-4-hydroxy-3-phthalidocyclopentene.

A method for preparing O-methoxy-carbonylbenzoylchloride is described byHoogwater et al., Rec. Trav. Chim. Pays-Bas, 92 (1973).

Preparation 6a dl-Trans-4-hydroxy-3-phthalimido-cyclopentene ##STR47##

A 31.3 g. (0.115 M) quantity of dl-trans-3-amino-4hydroxycyclopentenetoluenesulfonate is dissolved in 240 ml. of tetrahydrofuran and 47 ml.(0.46 M) of triethylamine. The resultant stirred solution is cooled to5° and treated dropwise with 22.8 g. (0.115 M) ofo-methoxycarbonylbenzoylchloride. The reaction is then allowed to warmto 25°. After 48 hours the reaction is partitioned between Ethyl acetateand water. The ethyl acetate layer is dried over magnesium sulfate andconcentrated in vacuo leaving 29.6 g. of residue. This residue ischromatographed over silica gel 60 eluted with 5 l. of (10-90) and 7.5l. of (20-80) acetone-methylene chloride. Three hundred ml. fractionsare collected. The product is isolated in fractions 25-35, which uponstanding crystallizes to yield 14.2 g. ofdl-trans:4-hydroxy-3-phthalido-cyclopentene, m.p. 114°-116°.

Preparation 6b dl-Trans-4-hydroxy-3-phthalimidocyclopentene ##STR48##

A quantity of crude dl-3,4-epoxycyclopentene is added to a suspension of7.2 g. (49 mM) of phthalimide and 1.43 g. (7.7 mM) of potassiumphthalimide in 23 ml. of dry dimethylformamide. After stirring at 25°for 114 hours the reaction is treated with 0.5 ml. (8.3 mM) of aceticacid and stirred for an additional hour. The reaction is then evaporatedin vacuo and the residue chromatographed over 300 g. of silica gel 60eluted with (10-90) acetone-CH₂ Cl₂. Fifty ml. fractions are collected.The product is collected in fractions 26-30, which upon standingcrystallizes to yield 0.58 g. ofdl-trans-hydroxy-3-phthalido-cyclopentene.

NMR (CDCl₃ δ): 2.1-3.4 (m, CH₂), 3.7 (OH), 4.6-5.0 (m, CHOH), 5.0-5.3(m, CHN), 5.5-6.2 (m, CH═CH), 7.7 (s, ArH).

TLC (silica gel 60): Rf=0.32 in (40-60) ethyl acetate-Skellysolve B andRf=0.71 in (15-85) acetone-CH₂ Cl₂.

Anal. Calc'd. for C₁₃ H₁₁ NO₃ : C, 68.14; H, 4.80. C, 68.19; H, 4.86.

Using the procedure of Preparation 6 but substituting the appropriatelysubstituted O-methoxy carbonylbenzoylchloride or 3-methoxy carbonylproponyl chloride for Omethoxy carbonylbenzoylchloride there is obtained

dl-trans-4-hydroxy-3-(3-nitrophthalimido)-cyclopentene

dl-trans-4-hydroxy-3-(4-methyl-phthalimido)-cyclopentene

dl-trans-4-hydroxy-3-succinimide-cyclopentene

dl-trans-4-hydroxy-3-(2,3-dimethylsuccinimidocyclopentene

dl-trans-4-hydroxy-3-hexahydrophthalimidocyclopentene

Preparation 6c ##STR49##

A 4.07 g. (17.67 mM) quantity ofdl-trans-4-hydroxy3-phthalido-cyclopentene is dissolved in 65 ml oftetrahydrofuran and 65 ml of ethyl alcohol. The solution is treated with0.94 ml (0.97 g., 19.44 mM) of hydrazine hydrate. After 10 minutes aprecipitate forms. After 2 hours the reaction is evaporated in vacuo(<25 mm and -30°). The residue is triturated with methylene chloride andfiltered to remove the insoluble phthalhydrazide. The filtrate isconcentrated in vacuo leaving dl-trans3-amino-4-hydroxy-cyclopentene asa solid.

Preparation 7 dl-Trans-3-amino-4-hydroxycyclopenteneN-trichloroethylurethane ##STR50##

A 9.9 g. quantity of crude dl-trans-3-amino-4-hydroxycyclopentene(prepared as in Preparation 3a) is dissolved in 75 ml. of water and themixture treated with 10.6 g. (100 mM) of sodium carbonate. The mixtureis then cooled in an ice bath to <10° and treated dropwise whilestirring vigorously, with 10.6 g. (50 mM) of trichloroethylchloroformateover 30 min. After 3 hours the reaction is acidified with coldconcentrated hydrochloric acid. The aqueous mixture is extracted threetimes with methylene chloride. The methylene chloride solution is washedwith water, dried over sodium sulfate and concentrated in vacuo leaving13.2 g. of brown oil. The oil is chromatographed over 750 g. of silicadioxide eluted with (10-90) acetone-methylene chloride. Fifty ml.fractions are collected. The product is found by TLC in fractions 55-85.Concentration of these fractions yields 5.0 g. oftrans-3-amino-4-hydroxy-cyclopentene-N-trichloroethyl urethane ascrystalline solid. m.p. 100°-103°.

NMR (CDCl₃, δ): 1.9-3.1 (m, CH₂), 4.0-4.65 (m, 3H), 4.74 (s, OCH₂),5.45-6.1 (m, CH=CH), 6.5-7.0 (NH).

TLC (silica gel 60): Rf=0.5 in (10-90) acetone-methylenechloride.

Preparation 7a dl-Trans-3-amino-4-hydroxycyclopentene-N-trichloroethylurethane ##STR51##

A 13.55 g. (50 mM) trans-3-amino-4-hydroxycyclopentene toluenesulfonateis dissolved in 75 ml. of water. The solution treated with 10.6 g. (10.0mM) of sodium carbonate, cooled in an ice bath, and treated dropwisewith 10.6 g. (50 mM) of trichloroethylchloroformate while stirringvigorously. After 75 min. the reaction is partitioned between water andmethylene chloride. The aqueous layer is separated and extracted twotimes more with methylene chloride. The combined methylene chloridelayer is dried over sodium sulfate and concentrated in vacuo. Crystalsof trans-3-amino-4-hydroxy-cyclopentene-N-trichloroethyl urethane areformed during the concentration. These are collected three times, eachtime rinsing the crystals with ether crop 1, 6.12 g. m.p. 105.5°-106°;crop 2, 4.35 g., m.p. 105-106; crop 3, 1.28 g., m.p. 104°-105°.

Using the same procedure as in Preparation 7 but substituting theappropriate halo-substituted alkylhaloformate or activated alkylcarbonate for trichloro ethyl formate there is obtained

dl-trans-3-amino-4-hydroxy cyclopentene-N-tbutyl urethane

dl-trans-3-amino-4-hydroxy cyclopentene-N-pmethoxybenzyl urethane

dl-trans-3-amino-4-hydroxy cyclopentene-N-diphenylmethyl urethane

dl-trans-3-amino-4-hydroxycyclopentene-N-[2iodoethyl urethane

Using the same procedure as in Preparation 7 but substitutingbenzyloxychloroformate there is obtaineddl-trans-3-(O-benzylcarbamoyl)-4-hydroxycyclopentene afterchromatography on silic gel with 85% methylene chloride/acetone.

NMR (CDCl₃)=2.1-2.9 (m, CH₂), 3.8-4.6 (m, 3H), 5.02 (5, OCH₂), 5.3-6.0(m, 3H), 7.22 (s, Ph).

TLC: Rf=0.58 (15% acetone/methylene chloride). Similarly prepared is theoptically active (d) trans-5(O-benzylcarbamoyl)-4-hydroxy cyclopenteneby substituting the d-startig material for the racemic.

Preparation 8 dl-2,2,2-Trichloroethylcis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate ##STR52##

An 11.75 g. (42.8 mM) quantity ofdl-trans-3-amino-4-hydroxy-cyclopentene-N-trichloroethyl urethane, a7.86 g. (48.3 mM) quantity of N-hydroxyphthalimide, and a 12.63 g. (48.3mM) quantity of triphenylphosphine are dissolved in 210 ml. oftetrahydrofurane dry. To the stirred solution is added dropwise over 15minutes 9.21 g. (53.1 mM) of diethylazodicarboxylates while holding thetemperature below 35° with an ice bath. The reaction is then stirred for1 hour at 25°, after which it is concentrated in vacuo (˜<25 mm. and˜30'). The residue is treated with ˜100 ml. of (10-60 ) ethylacetate-Skellysolve B and the precipitated triphenylphosphine oxideremoved by filtration after about 15 minutes. The residue is added tothe top of a 1 kg. silica gel 60 column which is eluted with 3 l. of(40-60) followed by (50-50) ethyl acetate-Skellysolve B. Three hundredml. fractions are collected. Fractions 14-19 are found to contain puredl-2,2,2-trichloroethyl cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamateby TLC. On concentration they yield 6.24 g. of crystals as a first crop,m.p. 138.5-139.5, 2.8 g. of a second crop m.p. 138.5°-139.5° and left2.13 g. of residue on evaporation to dryness. Fractions 20-24 are foundto contain by TLC a more polar impurity. These fractions are combinedwith the 2.13 g. of residue from above and rechromatographed over 750 g.of silica gel 6 q which is eluted with (10-90) ethyl acetate benzene.Three hundred ml. fractions are collected. Fractions 8-13 are found byTLC to contain product. Concentration of these fractions produced twomore crops of dl-2,2,2-trichloroethylcis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate crystals of 2.75 g. and2.62 g. with m.p.s. of 138.5°-139.5° and 137.5°-138.5°, respectively.Total yield 14.41 g.

NMR (CDCl₃, δ): 2.65-3.0 (m, CH₂), 4.77 (s, OCH₂) 4.7-5.2 (m, 2H),5.7-6.1 (m, CH═CH), 6.35-6.75 (NH), 7.83 (s, ArH).

TLC (silica gel 60): Rf=0.55 in (40-60) ethyl acetate Skellysolve B andRf=0.47 in (10-90) ethyl acetate-benzene.

Anal. Calc'd for C₁₀ H₁₃ Cl₃ N₂ O₅ : C, 45.79; H, 3.12; N, 6.68; Cl,25.35

Found: C, 45.92; H, 3.13; N, 6.62; Cl, 25.47.

Using the same procedure as in Preparation 8 but substituting theappropriate dl-trans-3-amino-4-hydroxycyclopentene-N-haloalkyl urethanefor dl-trans-3-amino-4-hydroxycyclopentene-N-trichloroethyl urethanethere is obtained

dl-t-butyl-cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate,

dl-p-methoxybenzyl-cis-5-(phthalimidoxy-2-cyclopentene-1-carbamate,

dl-diphenylmethyl-cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate,

Using the same procedure as in Preparation 8 but substituting theappropriate N-hydroxyphthalimides and N-hydroxysuccimides forN-hydroxyphthalimide there is obtained

dl-(2,2,2-trichloroethyl)-cis-5-(3-nitrophthalimido-oxy)-2-cyclopentene-1-carbamate,

dl-(2,2,2-trichloroethyl)-cis-5-(3-nitrophthal-imido)-2-cyclopentene-1-carbamate,

dl-(2,2,2-trichloroethyl)-cis-5-(succinimidoxy)-2-cyclopentene-1-carbamate,

dl-(2,2,2-trichloroethyl)-cis-5-(2,3-dimethyl-succinimidoxy)-2-cyclopentene-1-carbamate,

Using the same procedure as outlined in Preparation 8 but using variousalkyl methanes and substituted n-hydroxyphthalimides andn-hydroxysuccinimides there is obtained

dl-benzyl(cis-5-(3-nitrophthalimidoxy)-2-cyclopentene-1-carbamate

dl-p-methoxybenzyl-cis-5-(4-methylphthalimidoxy)-cyclopentene-1-carbamate.

Preparation 9 dl-Benzyl cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate##STR53##

A 1.46 g. (6.26 mM) quantity of trans-3-amino-cyclopentene-N-benzylurethane, a 1.12 g. (6.88 mM) quantity of N-hydroxyphthalimide, and 1.8g. (6.88 mM) of triphenylphosphine are dissolved in 25 ml. of drytetrahydrofuran. The solution is treated dropwise over about 5 min. with1.32 g. (7.6 mM) of diethyl azodicarboxylate in 5 ml. oftetrahydrofuran. The initial red solution after the start of theaddition turns yellow in about 10 minutes. After 16 hours the reactionis concentrated in vacuo (<25 mm at ˜30° ) and the residuechromatographed over 150 g. of silica gel. The column is eluted with 40ml. of (20-80) followed by (25-75) ethyl acetate benzene. Twenty-fiveml. fractions are collected. The product is found by TLC ion fractions13-20. Concentration of these fractions left 1.76 g. of di-benzylcis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate as an oil whichcrystallizes on standing. Recrystallization n from 50% ethylacetate/hexane gives m.p. 120°-2°.

NMR (CDCl₃, δ): 2.65-2.95 (m, CH₂), 4.8-5.10 (m, 2H), 5.15 (s, OCH₂),5.65-6.1 (m, CH═CH), 6.1-6.4 (NH), 7.37 (s, 5, ArH), 7.78 (s, 4, ArH).

TLC (silica gel 60): Rf=0.65 in (25-75)ethyl acetate benzene andRf=0.72in (5-95) acetone-methylene chloride.

Elemental Analysis for C₂₁ H₁₈ N₂ O₅

Calc'd: C, 66.65; H, 4.79; N, 7.41. Found: C, 66.16; H, 4.84; N, 7.32.

Using the same procedure, the d-benzyl-cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate is prepared by substituting thed-starting material for the racemic mixture.

Preparation 10 dl-2,2,2-Trichloroethylcis-5-(aminooxy)-2-cyclopentene-1-carbamate ##STR54##

A 7.42 g. (17.67 mM) quantity of dl-2,2,2-trichloroethyltrans-5-(phthalimidoxy)-2-cyclopentene-1-carbamate is dissolved in 65ml. of tetrahydrofuran and 65 ml. of ethyl alcohol. The solution istreated with 0.94 ml. (0.97 g., 19.44 mM) of hydrazine hydrate. After 10minutes a precipitate forms. After 2 hours the reaction is evaporated invacuo (<25 mn and ˜30° ). The residue is triturated with methylenechloride and filtered to remove the insoluble phthalhydrazide. Thefiltrate is concentrated in vacuo leaving 6.68 g. ofdl-2,2,2-trichloroethyl cis-5-(aminooxy)-2-cyclopentene-1-carbamate asan oil.

NMR (CDCl₃, ε): 2.35-2.65 (m, CH₂), 4.1-4.95 (m, 2H), 4.78 (s, OCH₂),5.35-6.1 (m, 5, NH, NH₂, (CH═CH).

TLC (silica gel 60): Rf=0.41 in (5-95) methanol-benzene and Rf=0.41 in(40-60) ethyl acetate Skellysolve B.

Using the same procedure as in Preparation 10, but substituting other dlhaloalkyl trans-5-(phthalimidoxy)-2-cyclopentene-1-carbamates fordl-2,2,2-trichloroethyltrans-5-(phthalimidoxy)-2-cyclopentene-1-carbamates there are obtainedcorresponding halo alkyl cis-5-(amino-oxy)-2-cyclopentene-1-carbamates.

Using the same procedure as in Preparation 10 but substituting dl ord-benzyl-cis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate there isobtained dl or d (resp.)benzyl-cis-5-(aminooxy)-2-cyclopentene-1-carbamate. The Rf=0.040 (50%ethyl acetate/hexane) (starting material Rf=0.71.

Preparation 11 dl-2,2,2-Trichloroethylcis-[[[(benzyloxy)carbonyl]amino]oxy]-2-cyclopentene-1-carbamate##STR55##

The crude dl-2,2,2-trichloroethyl cis-5-aminooxy-2-cyclopentene-1-carbamate (1.6 SmM) prepared in preparation 10 is dissolved in 10 ml. ofpyridine and the solution cooled in an ice bath and treated with 0.34 g(2.0 mM) of benzyloxychloroformate dissolved in 2 ml of CH₂ Cl₂. After1.5 hours the reaction mixture is poured into water and methylenechloride and treated with (ammonium chloride until acidic). Themethylene chloride layer is separated, dried over sodium sulfate andconcentrated in vacuo leaving 766 mg of oil. The oil is chromatographedover 55 g of silica gel 60, eluted with (40-60) ethylacetate-Skellysolve B. Then ml fractions are collected. Fractions 10-13,which are shown by TLC to contain the product, are combined andconcentrated in vacuo leaving 535 mg of residue. After addition ofisopropylether to the residue and scratching, the material crystallizedto yield dl-2,2,2-trichloroethylcis-[[[(benzyloxy)carbonyl]amino]oxy]-2-cyclpentene-1-carbamate, m.p.86-89° C.

NMR (CDCl₃, ε): 2.2-2.9 (m, 2H), 4.2-5.0 (m, CHN, CHO), 4.67 (s, OCH₂CCl₃), 5.16 (s, OCH₂ φ), 5.4-6.2 (m, CH═CH), 7.37 (s, φ), 7.6-8.0 (m,NH), 8.47 (s, NH).

TLC (silica gel 60): Rf=0.73 in (40-60) ethylacetate-Skellysolve B.

Using the same procedure as in Preparation 11 but substituting either dlor (IR,5S)-benzyl-cis-5-(aminooxy)-2-cyclopentene-1-carbonate for llldand 2,2,2-trichloroethylchloroformate for benzyloxychloroformate thereis obtained dl or d (resp.) benzyl-cis[([2,2,2-trichloroethoxy)carbonyl)aminooxy]-2-cyclopentene-1-carbamate.

NMR (CDCl3): 7.30 (s,pH), 5.6-61 (m, 2H), 5.10 (5, OCH₂), 44-5.0 (m,2H), 4.72 (s, CCl₃ CH₂), 2.4-2.8 (m, 2H).

TLC: Rf=0.83 (50% ethyl.acetate/hexane)

Preparation 12 dl-Benzyl cis-[(2-amino-3-cyclopenten-1-yl)oxy]carbamate##STR56##

(a) Zinc, Ammonium Chloride, Methanol Procedure

A 21.32 g (50.4 mM) quantity of dl-2,2,2-trichloroethylcis-[[[(benzyloxy)carbonyl]amino]oxy]-2-cyclopentene-1-carbonate isdissolved in 425 ml of methanol and the solution treated with 21.3 g(326 mM) of zinc and 10.7 g (200 mM) of ammonium chloride. The resultantmixture is stirred vigorously for 60 minutes at which time TLC showedthe reaction to be complete. The reaction is mildly exothermic and onthe scale described warmed from a temperature of 25° at the start of thereaction to a maximum of 35° C. The reaction mixture is then filteredand the filtrate concentrated in vacuo. The filtered solids are washedwith about 200 ml of 5% sodium bisulfate (aqueous) and this washing isadded to the residue from evaporation of the methanol. The resultantaqueous mixture is partitioned between methylene chloride and themethylene chloride layer is separated. The aqueous layer is made basicto pH8 with conc. ammonium hydroxide and then is treated with 4 g ofsodium cyanide and the resultant solution extracted 3 times with ˜200 mlportions of methylene chloride. The combined methylene chloridesolutions are dried over sodium sulfate and concentrated in vacuo toyield 9.6 g (77%) of benzylcis-[(2-amino-3-cyclopenten-1-yl)oxy]carbamate as an oil.

NMR (CDCl₃, δ): 2.2-2.7 (m, 2H), 3.6-5.5 (m), 5.08 (s, OCH₂), 5.5-5.9(m, CH═CH), 7.28 (s, 5Ar-H).

TLC (silica gel 60): Rf=0.42 in (1-10-90) ammoniumhydroxide-methanol-methylene chloride.

The reaction produces zinc complexes of product dl-benzylcis-[(2-amino-3-cyclopenten-1-yl) oxy]carbamate which are insoluble inMeOH and most other organic solvents. These can be dissolved in aqueousacid.

The compound under basic pH makes strong complexes with zinc. Theproduct can be extracted from aqueous only if the complexes are brokenup. EDTA tetra sodium salt also works but not as well as sodium cyanide.

(b) Zinc, Ammonium Chloride, Methanol, Sodium Hydroxide Workyp.

The zinc, ammonium chloride reduction in methanol is run as above inprocedure (a) on a 17.67 mM quantity of dl-2,2,2-trichloroethylcis-[[[(benzyloxy)carbonyl]amino]-oxy]-2-cyclopentene-1-carbamate. Afterthe extraction of the acidic aqueous solution to remove dichloroby-product, the aqueous is made basic to pH 14 with 50% sodium hydroxide(using ice to keep the mixture cool). At pH ˜8-10a volumous preciptateof zinc hydroxide forms but this redissolves at higher pH leaving thezinc salt of the desired product. This salt is insoluble in mostsolvents except for aqueous acid. The resultant precipitate is filteredand dried. This zinc salt of dl-benzyl cis[(2-amino-3-cyclopenten-1-yl)oxy]carbamate is dissolved in the minimumamount of ammonium chloride to effect solution. This is thenfreeze-dried and the residue used in Preparation 13a.

(c) Zinc, Methane Sulfonic Acid, Methanol Procedure

A 10 g. (23.6 mM) quantity of dl-2,2,2-trichloroethyl cis[[[((benzyloxy)carbonyl]amino]oxy-2-cyclopentene-1-carbamate isdissolved in 200 ml. of methanol. The solution is treated with 10 g.(154 mM) of zinc dust. To this mixture is added over 25 minutes, whilestirring vigorously. 4 ml. of methanesulfonic acid. TLC shows thereaction to be complete in less than 1 hour. The reaction mixture isthen filtered and the zinc solids washed with methanol. The filtrate andcombined washings are concentrated in vacuo and the residue is useddirectly in Preparation 13.

(d) Zinc, Acetic Acid, Water Procedure.

A 1.0 g. (2.36 mM) quantity of dl-2,2,2-trichloroethyl cis[[[(benzyloxy)carbonyl]amino]oxy]cyclopetene-1-carbamate is dissolved in10 ml of (9-1) acetic-acid water and the solution treated with a totalof 1.0 g (15.45 mM) of zinc added in 5 equal portions in 45 minintervals. After a total of 6 hours from the initial addition, TLC showsthe reaction to be complete. The reaction mixture is then filtered andthe filtrate partitioned between 5% sodium bisulfate and methylenechloride. The aqueous layer is separated and made basic to pH 9-10 withconc. ammonium hydroxide using ice to keep the mixture cool. Theresultant aqueous solution is extracted three times with methylenechloride. The combined methylene chloride solutions are dried oversodium sulfate and concentrated in vacuo leaving 516 mg (88%) ofdl-benzyl cis-[(2-amino-3-cyclopenten-1-yl)oxy]carbamate as an oil.

In the acetic acid runs, NH₄ OH seems to be adequate to break up thezinc salts. NH₄ OH is not sufficient for the products from procedures B12(a) and 12(c).

Preparation 13dl-Benzyl-cis-[(2-phthalimido-3-(cyclopenten-1-yl)oxy]carbamate (30)##STR57## (a) An 11.0 g. (46.6 mM) quantity of crudebenzyl-cis-(]2-amino-3-cyclopenten-1-yl)oxy]carbamate from step 34procedure (a) is dissolved in 250 ml of tetrahydrofuran. To this isadded 11.5 g (58.3 mM) of 2-methoxy carbonylbenzoyl chloride solved in250 ml of tetrahydrofuran. To this is added 11.5 g (58.3 mM) of2-methoxy carbonylbenzoyl chloride and 31 ml (23.5 g, 232 mM) oftriethylamine. The reaction is warmed to 50° for 72 hours at which timeTLC indicates the reaction has gone to completion. The reaction is thenconcentrated in vacuo. The residue is partitioned between ethyl acetateand ammonium chloride. The ethyl acetate layer is separated and washedwith 5% sodium bisulfate and water and dried over magnesium sulfate.Concentration of the ethyl acetate solution in vacuo leaves 22.9 g ofcrude residue. The residue is chromatographed over 2 kg of silica gel 60eluted with (40-60) ethyl acetate-Skellysolve B. Four hundred mlfractions are collected after taking a forerun of 2 l. The product isfound in lr 16-24 by TLC. Concentration of these fractions gives 13.15 g(75%) of dl cis-[2-phthalimido-3-(cyclopenten-yl)oxy]carbamate.

NMR (CDCl₃, ε): 2.4-3.2 (m, CH₂), 4.5-5.5 (m, 4), 5.00 (s, OCH₂),5.5-6.2 (m, CH═CH), 7.27 (s, 5ArH), 7.5-8.0 (m, 4ArH),

TLC (silica gel 60)Rf=0.48 in (40-60) ethyl acetate-Skellysolve B.

(b) Using the same procedure as in (a) above or 17.67 the crudedl-benzyl-cis-[(2-amino-3-cyclopenten-1-yl)oxy]-carbamate Zn salts, a3.23 g. (48%) yield ofcis-[2-phthalimido-3-cyclopenten-1-yl)oxy]carbamate is obtained. (c)Using the same procedure as in (a) and (b) above on 23.6 mM ofdl-benzyl-cis-[(2-amino-3-cyclopenten-1-yl)-oxy]carbamate, a 6.91 g.(77%) yield of dl-cis[(2-phthalimido-3-cyclopenten-1-yl)oxy]carbamate isobtained.

The combined steps 12(c) and 13(c) are the preferred method forconverting dl-2,2,2-trichloroethylcis-[[[(benzyloxy)carbonyl]amino]oxy]-2-cyclopentene1-carbamate todl-benzyl-cis-[2-phthalimido-3-(cyclopenten-1-yl)oxy]carbamate.

In addition, both dl- andd-cis-(2-phthalimido-3-[cyclopenten-1-(2,2,2-trichloroethoxycarbonyl)aminooxy]-carbamatecan be prepared by using Preparation 13 but substituting dl- ord-2,2,2-trichloroethyl-cis-[(2-amino-3-cyclopenten-1-yl]oxy]carbamate.This substituted starting material can be prepared by treating dl or d(resp) -benzyl-cis[(]2,2,2-trichloroethoxy]carbonyl)aminooxy]-2-cyclopentene-1-carbamate(prepared by Preparation 11) with HBr saturated glacial acetic acid forone hour at room temperature. The reaction solution is concentrated invacuo to afford the product which can be used directly as above inPreparation 13.

NMR (CDCl₃): 7.5-8.0 (m, 4H), 8.45 (m, NH), 5.5-6.2 (m, 2H), 4.35-5.5(m, 4H), 4.69 (s, CCl₃ CH₂), 2.6-3.0 (m, 2H).

TLC: RF=6.67 (50% ethylacetate/hexane)

Preparation 14 (αS,5S andαR,5R)-2-[(benzyloxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazolidine-aceticacid ##STR58## An 10.0 g (26.5 mM) quantity ofdl-cis-[2-phthalimido-3-cyclopenten-1-yl)oxy]carbamate llla is dissolvedin 250 ml of acetone and 180 ml of water. The resultant solution istreated with 28 g (130 mM) of sodium iodate and 100 mg of rutheniumchloride hydrate (1-3 H₂ O) in 5 ml. of water. The reaction is stirredvigorously for one hour. After about the first 15 minutes a slightexotherm occurs causing the temperature to rise from about roomtemperature (˜23° ) to about 35°. The reaction is then evaporated at <30mm and <30° C. to remove acetone. The aqueous residue is partitionedbetween ethyl acetate and water acidified with ˜10 ml of M sulfuricacid. The aqueous layer is separated and extracted 2 times more withethylacetate. The combined ethylacetate solutions are washed withsaturated sodium chloride solution, dried over magnesium sulfate andconcentrated in vacuo (<30 mm, < 30° C.) leaving a glassy foam. Theresidue is chromatographed over 1 kg of CC-4 silica gel eluted with alinear gradient prepared from 5 l of (40-60) ethyl acetate-Skellysolve Band 5 l of (80-20) ethyl acetate-Skellysolve B. Fractions (400 ml) 16-19yield 7.48 g. of essentially pure (α5,5S and2R,5R)-2-[(benzyloxy)-carbonyl]-3-oxo-α-phthalimido-5-isoxazoline aceticacid, m.p. 113-116° (from ethanol).

NMR d₈ -acetone, ε): 3.16 (d, J=7.5, CH₂), 5.25 (s, OCH₂), 5.1-5.7 (m,2), 7.39 (s, 5), 7.92 (s, 4).

Elemental Analysis: Calc'd for C₂₁ H₁₆ N₂ O₈

Calc'd: C, 59.43; H, 3.80; N, 6.60.

Found: C, 58.79; H, 3.80; N, 6.53.

Fraction 14 yields 0.93 g of2-[(Benzyloxy)-carbonyl]-3-oxo-4-phthalimido-5-isoxazolidine aceticacid, m.p. 160-2° (ethyl acetate/hexane, 1:1).

NMR d₆ -acetone, ε): 3.06 (d, J=5.5, CH₂), 5.38 (s, OCH₂), 5.1-5.8 (m,2), 7.45 (m, 5) 7.88 (s, 4).

Elemental Analysis: Calc'd for C₂₁ H₁₆ N₂ O₈.H₂ O

Calc'd: C, 57.01; H, 4.10; N, 6.33.

Found: C, 57.24; H, 3.98; N, 6.45.

A mixture of the two acids is found in Fraction 15.

Using the same procedure as in Preparation 14 but substituting dl- ord-cis-(2-phthalimido)-3[cyclopententene-1-(2,2,2-trichloroethoxycarbony)aminooxy]carbamateaffords (α5,5S andαR,5R)-2-](2,2,2-trichloroethoxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazolidine-aceticacid (57% yield), m.p. 208-210°.

NMR (acetone-d₈): 3.27 (d, J=7.5 Hz, CH₂), 4.92 (s, CCl₃ CH₂), 5.1-5.7(m, 2H), 7.91 (s, 4H).

TLC: Rf=0.41 (A lX)

Analysis: Calc'd for C₁₀ H₁₁ Cl₃ N₂ O₈

Calc'd: C, 41.27; H, 2.38; N, 6.02.

Found: C, 41.33; H, 2.36; N, 6.04.

The isomeric (4S,5S and4R,5R)-2-[(2,2,2-trichloroethoxy)carbonyl]-3-oxo-4-phthalimido-5-isoxazolidineacetic acid is also produced in 22% yield.

NMR (acetone-d₈): 3.10 (d, J=5.5 H₃, 2H), 5.08 (5, CCl₃ CH₂), 5.1-5.8(m, 2H), 7.90 (s, 4H).

TLC: Rf=6.52

Preparation 15 (α5,5S and αR,5R)-3-Oxo-α-phthalimido-5-isoxazolidineacetic acid ##STR59##

A 7.48 g 17.6 mM) quantity of (αS,5S andαR,5R)-2-[(benzyloxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazolidineacetic acid is dissolved in 150 ml. of ethyl acetate and 75 ml. of 95%ethanol. The solution is treated with 1.5 g. of palladium black andhydrogenated at 25° C. and 1 atmosphere of pressure. The reaction isstopped after 165 minutes, filtered and the filtrate used directly forthe preparation of (αS,5S and αR,5R)-3-oxo-α-phthalimido-5-isoxazolidineacetic acid.

Using the same procedure as above in a different run the filtrate isevaporated in vacuo to yield (αS,5S and αR,5R)-3-oxo-α-phthalimido-5-isoxazolidine acetic acid as a residue.

NMR (CD₃ OD, ε): 2.92 (partially split d, CH₂), 5.1-5.7 (m, 2), 7.85 (s,4 ArH).

TLC (silica gel 60): Rf=0.36 in the upper phase of (9-2-5-10)ethylacetate-acetic acid cyclohexanewater. In the same system thestarting material Rf=0.46

Using the same procedure as in Preparation 15 with dl or dVllld affordsthe dl or d (resp.) isotricholomic acid phthalimide Vllc as a white foamwith TLC R_(f) =0.54 (starting material R_(f) =0.64)

NMR (Acetone-d₆): 9.1 (s, CO₂ H), 7.83 (s, 4H), 5.0-5.8 (m, 2H), 2.95(m, 2H).

Alternatively, the benzyloxycarbonyl group can be removed from llc toproduce llb (or Vlld to Vllc) with acid. For example, a 424 mg (1 mmole)amount of llb is dissolved in 5 ml of glacial acetic acid under N₂ anddry HBr is bubbled into the reaction solution for 10 minutes. Afterstirring for 4 hours at room temperature the solution is concentratedover chromatographed on 50 g of CC-4 silica gel with 70%ethylacetate/hexane. This procedure yielded 170 mg (59%) of productidentical to that produced hydrogenolytically (by NMR and TLC). Otheracids and solvents such as HBr/CH₃ NO₂, HBR/CH₂ Cl₂ and trifluoroaceticacid can also be employed.

Preparation 16 (αS,5S and αR,5R(-3-oxo-α-phthalimido-5-isoxazolidineacetic acid, benzhydryl ester ##STR60##

A 5.2 g (26.5 mM) quantity of benzophenone hydrazone was dissolved in125 ml of ethyl ether. The solution is treated with 10 g of sodiumsulfate, 11.1 g (51 mM) of yellow mercury oxide and 0.2 ml of asaturated potassium hydroxide solution in ethyl alcohol. The reaction isstirred 60 minutes and the resultant deep burgandy mixture filtered. Theresultant solution is added directly to the reaction solution from a17.6 mM hydrogenation in which (αS,5S andαR,5R)-3-oxo-α-phthalimide-5-isoxazolidine acetic acid is prepared. TLCshows the reaction to be finished in less than one hour. The reaction isthen treated with sufficient 3 N hydrochloric acid while stirringvigorously, to destroy the excess burgundy colored diazo compounds. Thereaction mixture is then evaporated in vacuo (<30 mm and <30° C.) andchromatographed over 500 g. of CC-4 silica gel eluted with (40-60) ethylacetate-toluene. Two hundred ml. fractions are collected. The (αS,5S andαR,5R)-3-oxo-phthalimido-5-isoxazolidine-acetic acid, benzhydryl esteris found in fr 10-14 (3.05 g., 38% yield).

NMR (CDCl₃ ε): 2.75 (d, J=11.5, CH₂), 5.15-5.7 (m, 2), 6.93 (s, CH (C₆H₅)₂), 7.18 (s, C₆ H₅), 7.28 (s, C₆ H₅), 7.5-8.0 (m, 4, ArH).

TLC (silica gel 60): Rf=0.42 in (40-60) ethyl acetatetoluene. The isoproduct from compound 33 has Rf=0.59 in this same system

Utilizing the procedure similar to the procedure of Preparation 16, butsubstituting RN₂ for diphenyldiazomethane, where R can be methyl, ethyl,benzyl and the like, produces the corresponding esters of IIb. Withthese more reactive alkyl diazo compounds it is more efficient to treatthe acid, IIb, in a non-protic solvent, such as tetrahydrofuran, inplace of ethanol.

Alternatively, esters of IIb can be produced by alkylation of the acidin base with a reactive alkylating agent. For example, a 87 mg (0.3mmole) quantity of IIb in 3 ml of dry acetonitrile under nitrogen istreated with 50 μl of diisopropylethylamine and 60 mg of p-methoxybenzylbromide. After 16 hours the solution is distributed between ethylacetate/water, and the organic phase separated dried over sodium sulfateand concentrated. Preparative TLC (50% ethyl acetate/hexane) affords 55mg (45% of the p-methoxybenzyl ester.

NMR (CDCl₃): 7.81 (m, 4H), 7.05 (A₂ B₂,4H), 5.1-5.7 (, 2H), 5.13 (s,2H), 3.76 (s, 3H, CH₃ O), 2.81 (d, J=7.5 H₃, 2H).

Utilizing procedures detailed above but substituting the dl- ord-isotrichlomic acid phthalmide VIIc for IIc affords dl-d(respectively)-VIIIb, isotrichlomic phthalimide ester.

Preparation 17dl-Trans-3-phthalimido-4-fluoromethanesulfonyloxycyclopentene ##STR61##

A 5 g (21.8 mM) quantity of 4-hydroxy-3-phthalimidocyclopentene and 1.72g (21.8 mM) of pyridine are dissolved in 6 ml of methylene chloride andthe solution added dropwise over 40 minutes to an ice-cooled stirredsolution of 4.6 g (21.8 mM) of trifluoromethanesulfonic anhydride in 15ml of methylene chloride under nitrogen. The solution is stirred for anadditional 15 minutes and then washed with water and dried concentrationof the methylene chloride solution leavesdl-trans-3-phthalimido-4-trifluoromethanesulfonyloxycyclopentene.

Preparation 18dl-Benzyl(cis-[(2-phthalimido-3-cyclopentene-yl)oxy]carbamate ##STR62##

A 21.8 mM quantity ofdl-trans-3-phthalimido-4-trifluoromethanesulfonyloxycyclopentene isdissolved in 50 ml of methylene chloride and the solution treated with21.8 mM of the potassium salt of N-hydroxy-benzylurethane. Afterstirring 24 hours under nitrogen the reaction is extracted with 5%aqueous sodium bicarbonate and water and dried. Distillation of themethane chloride leaves a residue which is chromatographed over silicagel, eluted with (40-60) ethylacetate-Skellysolve B. After concentrationof the fraction found by TLC, to contain product, there is obtainedd-benyl-cis-[(2-phthalimido3-cyclopentene-yl)oxy]carbamate.

PREPARATION OF OPTICALLY ACTIVE ISOMERS

Preparation 17 Resolution dl-trans-3-amino-4-hydroxycyclopentene (5):Preparation of (+)-deoxycholate 6b and (-) tartarate 6c ##STR63##

(1) A 49.5 g (0.50 M) quantity of dl trans-3-amino-hydroxycyclopentenedissolved in about 100-200 ml of methanol and 98 g (0.25 M) ofdeoxycholic acid dissolved in about 200-300 ml of methanol are mixed andallowed to crystallize for several hours. The crystals oftrans-3-amino-4-hydroxycyclopentene are collected by filtration andwashed three times with some portions of methanol. There is obtained 105g of trans-3-amino-4-hydroxycyclopentene deoxycholate as light beigecrystals, m.p. 195°-197° (dec). The mother liquors are saved for Step 2.

(2) A 500 ml quantity of Amberlite IRA-400 (Cl⁻ form) is added to a 600ml fritted funnel and washed six times with 200 ml portions of N sodiumhydrochloride, 3 times with water (3×200 ml), with 200 ml portions ofwater three times with methanol. A 250 ml quantity of this hydroxy resinand the mother liquors from Step 1 above are mixed and stirred for 2hours under nitrogen. The mixture is then poured on top of the other 250ml of resin packed into a chromatography column in methanol. The columnis eluted with methanol collecting 250 ml fractions. The product isfound by TLC in fractions 106. Evaporation of these fractions leaves 32g of (3R,4R)-3-amino-4-hydroxycyclopentene.

TLC (silica gel 60): Rf=0.24 in (1-20-80) ammonium hydroxide-methanolCH₂ Cl₂. Detected by potassium permanganate spray.

(3) The residue from Step 2 above is dissolved in 300 ml. of 95% ethanoland the solution mixed with a solution of 37.5 g (0.25 M) ofL-(+)-tartaric acid dissolved in 300 ml. of 95% ethanol. The mixedsolutions are seeded with previously resolved salt. After 2-3 hours, thecrystals are collected by filtration and dried. There is thus obtained54.4 g of crystals, m.p. 84°-86°. The crystals are recrystallized from700 ml of 95% ethanol. There is thus obtained 48.5 g of crystalline(3R,4R)-3-amino-4-hydroxycyclopentene L(+)-tartaric acid salt. m.p.86.5°-88.5° [α]₅₇₈ ²⁰ =-37.8° (c=3.20 in 4). A 200 ml quantity ofAmberlite IRA-- 400 (OH⁻ form) and 300 mloof methanol was added to 25 gof resolved tartarate salt from step 3. The mixture is stirred under anitrogen atmosphere for 1 hours. Another 100 ml of Amberlite IRA-400(OH⁻ form) is added to a chromatography column and the above mixture oftartarate salt, Amberlite resin and supernatant methanol solution addedto the top. The methanol is drained from the column and fresh methanolis passed through until a total of 1500 ml of methanol is collected. Themethanol solution is then evaporated in vacuo leaving 10.18 g ofcrystalline residue (3R,4R)-3-amino-4-hydroxycyclopentene, m.p. 67°-75°[α]₅₇₈ ²⁰ =-139° (c=1.0, MeOH).

Preparation 20 (1R,5R)-2,2,2-trichloroethyl)-5-hydroxy2-cyclopentene-1-carbamate ##STR64##

A 10.18 (103 mM) quantity of crude (3R,4R)-3-amino-4-hydroxycyclopentene (prepared as in Preparation 19, step4) is dissolved in 75 ml of water and the mixture treated with 10.6 g(100 mM) of sodium carbonate. The mixture is then cooled in an ice bathto <10° and treated dropwise while stirring vigorously, with 24 g (110mM) of trichloroethylchloroformate over 30 minutes. After 3 hours thereaction is filtered and the collected solid product washed throughlywith water. On drying there is obtained 22.43 g of (1R,5R)-2,2,2-trichloroethyl-5-hydroxy-2-cyclopentene-1-carbamate ascrystalline solid, m.p. 82°-84°. Recrystallization of a small samplefrom isopropylether-Skellysolve B gives material with a m.p. 87°-87.5°[α]-92° (C=0.14, MeOH).

TLC (silica gel 60): Rf=0.5 in (10-90) acetone-methylenechloride.

Preparation 21(IR,5S)-2,2,2-trichloroethyl-5-(phthalimidoxy)-2-cyclopentene-1-carbamate##STR65##

A 21 g (76.5 mM) quantity of(1R,5R)-2,2,2-trichloroethyl-5-hydroxy-2-cyclopentene-1-carbamate, a13.76 g. (84.4 mM) quantity of N-hydroxyphthalimide, and a 22.1 g. (84.4mM) quantity of triphenylphosphine are dissolved in 400 ml. oftetrahydrofurane dry. To the stirred solution is added dropwise over 15minutes 16.12 g. (92.6 mM) of diethylazodicarboxylates while holding thetemperature below 35° with an ice bath. The reaction is then stirred for1 hour at 25°, after which it is concentrated in vacuo (˜<25 mm. and˜30°). The residue is treated with ˜100 ml. of (10-60) ethylacetate-Skellysolve B and the precipitated triphenylphosphine oxideremoved by filtration after about 15 minutes. The residue is added tothe top of a 2 kg. silica gel 60 column which is eluted with 3 l. of(40-60) followed by (50-50) ethyl acetate-Skellysolve B. Three hundredml. fractions are collected. Fractions 19-27 are found to contain pure(IR,5S)-2,2,2-trichloroethylcis-5-(phthalimidoxy)-2-cyclopentene-1-carbamate by TLC. Onconcentration they yield 26.9 g. of(IR,5S)-2,2,2trichloroethyl-5-(phthalimidoxy)-2-cyclopentene-1carbamatecrystals, 117°-118° [α]_(o) =-26° (C=0.56, MeOh).

Preparation 22(IR,5S)-2,2,2-trichloroethyl-5-(aminooxy)-2-cyclopentene-1-carbamate##STR66##

A 24.9 g. (17.67 mM) quantity of(IR,5S)-2,2,2-trichloroethyl-5-(phthalimidoxy)-2-cyclopentene-1carbamateis dissolved in 230 ml. of tetrahydrofuran and 230 ml. of ethyl alcohol.The solution is treated with 3.3 ml. (3.4 g., 68 mM) of hydrazinehydrate. After 10 minutes a precipitate forms. After 2 hours thereaction is evaporated in vacuo (<25 mn and ˜30°). The residue istriturated with methylene chloride and filtered to remove the insolublephthalhydrazide. The filtrate is concentrated in vacuo leaving 23.3 g.of a semisolid. This material is triturated with 100 ml of ethyl acetateand filtered. The filtered solids are washed with 100 ml of methylenechloride. The combined filtrates are evaporated in vacuo leaving 18.51 gof (IR,5S)-2,2,2-trichloroethyl5-(aminooxy)-2-cyclopentene-1-carbamateas an oil.

TLC (silica gel 60): Rf=0.41 in (5-95) methanol-benzene and Rf=0.41 in(40-60) ethyl acetate Skellysolve B.

Using the same procedure as in Preparation 23, but substituting theappropriate haloalkyl(IR,5S)-5-(phthalimidoxy)-2-cyclopentene-1-carbamate for(IR,5S)-2,2,2-trichloroethyl-5-(phthalimidoxy)-2-cyclopentene-1-carbamatesthere is obtained the correspondinghaloalkyl-5-(aminoxy)-2-cyclopentene-1-carbamates.

Preparation 23(IR,5S)-2,2,2-trichloroethyl-5-[[[(benzyloxy)carbonyl]amino]oxy]-2-cyclopentene-1-carbamate##STR67##

An 18.5 g. quantity of crude(IR,5S)-2,2,2-trichloroethyl-5-aminooxy-2-cyclopentene-1-carbamateprepared as in preparation 10 is dissolved in 175 ml. of pyridine andthe solution cooled in an ice bath and treated with 11.6 g. (67.7 mM) ofbenzyloxychloroformate dissolved in 15 ml. of methylene chloride. After1.5 hours the reaction mixture is treated with 10 ml. of water, stirred10 minutes and the reaction mixture is poured into water and methylenechloride and treated with ammonium chloride until acidic. The methylenechloride layer is separated, dried over sodium sulfate and concentratedin vacuo leav- 22.6 g. of oil. The oil is crystallized from ether giving5.2 g. of(IR,5S)-2,2,2-trichloroethyl[((benzyloxy)carbonyl)amino)oxo]-2-cyclopentene-1-carbamate,m.p. 89.5-90.5. A second crop of 9.17 g., m.p. 89-90 and a third crop of1.54 g., m.p. 80-85 is obtained by crystallization of the first cropmother liquors from isopropyl ether. [α]_(D) =-33° (C=0.02, MeOH).

TLC (silica gel 60): Rf=0.73 in (40-60) ethylacetateSkellysolve B.

Preparation 23a(IR,5S)-2,2,2-trichloroethyl-5-[[[(tbutyloxy)carbonyl]amino]oxy]-2-cyclopentene-1-carbamate

An 7.8 g. quantity of crude(IR,5S)-2,2,2-trichloroethyl-5-(aminooxy)-2-cyclopentene-1-carbamate isdissolved in 25 ml. of tetrahydrofuran and 6.15 g (25 mm) of2-(tertbutoxycarbonyloxyimino)-2-phenylacetonitrile and the mixturestirred for 22 hours at room temperature, 20 hours at 50° C., 72 hoursat room temperature and then evaporated in vacuo. The residue ischromatographed over 900 g. of silica gel eluted with (15-85)ethylacetate-toluene. Three hundred ml. fractions are collected. Theproduct is found in fractions 10-12. Evaporation and recrystallizationof the residue from Skellysolve B yields(IR,5S)-2,2,2-trichloroethyl-5-[[[(t-butyloxy)carbonyl]amino]oxy]-2-cyclopentene1-carbamatem.p. 83°-84° C. as a white solid.

NMR (CDCl₃, ε): 1.45 (s,9) 2.45=2.7 (m,2), 4.3-4.9 (m, 2) 4.75 (s, 2),5.6-6.1 (m, 3H), 7.7-7.8 (b,1).

Preparation 24 (1S,2R)-benzyl-[(2-amino-3-cyclopenten1-yl)oxy]carbamate.##STR68##

A 7.12 g. (16.8 mM) quantity of(IR,5S)-2,2,2-trichloroethyl-5-[[[(benzyloxy)carbonyl]amino]oxy-2-cyclopentene-1-carbamateis dissolved in 75 ml. of methanol. The solution is treated with 7 g.(108 mM) of zinc dust. To this mixture is added over 10 minutes, whilestirring vigorously, 2.1 ml. of methanesulfonic acid. TLC shows thereaction to be complete in less than 90 minutes. The reaction mixture isthen filtered and the zinc solids washed with methanol. The filtrate andcombined washings are concentrated in vacuo leaving 5.8 g of crude(1S,2R) benzyl-[(2-amino-3-cyclopenten-1-yl)oxy]carbamate which is useddirectly in Preparation 23.

Using the same procedure as in Preparation 24 but substituting(1R,5S)-2,2,2-trichloroethyl-5-[[[(t-butyloxy)carbonyl]amino]oxy-2-cyclopentene-1-carbamatefor(1R,5S)-2,2,2-trichloroethyl-5-[[[(benzyloxy)carbonyl]amino]oxy-2-cyclopentene-1-carbamatethere is obtained(1S,2R)-t-butyl-5-[(2-amino-3-cyclopentne-1-yl)oxy]carbamate.

Preparation 25(1S,2R)-benzyl-(2-phthalimido-3-(cyclopenten-1-yl)oxy]carbamate##STR69##

A 5.8 g. (16.8 mM) quantity of crude (1R,5R)-benzyl[(2-amino-3-cyclopentene-1-yl)oxy]carbamate from Preparation 23 isdissolved in 90 ml. of tetrahydrofuran. To this is added 4.95 g (25 mM)of 2methoxy carbonylbenzoyl chloride and 10 ml. (7.3 g., 72 mM) oftriethylamine. The reaction is warmed to 50° for 72 hours at which timeTLC indicates the reaction has gone to completion. The reaction is thenconcentrated in vacuo. The reaction is partitioned between ethyl acetateand ammonium chloride. The ethyl acetate layer is separated and washedwith 5% sodium bisulfate and water and dried over magnesium sulfate.Concentration of the ethyl acetate solution in vacuo leaves 7.4 g. ofcrude residue. The reaction is chromatographed over 600 g. of silicagel, 60 eluted with (40-60) ethyl acetateSkellysolve B. Fifty ml.fractions are collected. The product is found in fr 16-24 by TLC.Concentration of these fractions gives 5.2 g. (75%) of(1S,2R)-benzyl[2-phthalamido-3-(cyclopentene-yl)oxy]carbamate as acrystalline solid, m.p. 89-92 [α]_(D) =-143 (C=0.7, MeOH).

TLC (silica gel 60): Rf=0.48 in (40-60) ethyl acetateSkellysolve B.

Preparation 26(αS,5S)-2-[(benzyloxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazolidineaceticacid ##STR70## An 10.0 g (26.5 mM) quantity of (2R,3S)-benzyl-[2phthalimido-3-cyclopenten-1-yl)oxy]carbamate IIIa isdissolved in 250 ml. of acetone and 180 ml. of water. The resultantsolution is treated with 28 g. (130 mM) of sodium iodate and 100 mg. ofruthenium chloride hydrate (1-3 H₂ O) in 5 ml. of water. The reaction isstirred vigorously for one hour. The reaction is then evaporated at <30mm and <30° C. to remove acetone. The aqueous residue is partitionedbetween ethyl acetate and water acidified with ˜10 ml of M sulfuricacid. The aqueous layer is separated and extracted two times more withethylacetate. The combined ethyl acetate solutions are washed withsaturated sodium chloride solution, dried over magnesium sulfate andconcentrated in vacuo (<30 mm, <30° C.) leaving a glassy foam. Theresidue is added to the top of a 1 kg. of CC-4 silica gel column on 80g. of CC-4 silica gel column on which it has been deposited byevaporation of a methylene chloride solution and eluted. Four hundred mlfractions are collected. Evaporation of fractions 16-19 yields, 7.48 g.of essentially pure(αS,5S)-2-[(benzyloxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazoline aceticacid, m.p. 113-116° [α]_(D) =-57° (C=2.0 in MeOH).

Fraction 14 yields 0.93 g. of (45,5S)2-[(benzyloxy)carbonyl]-3-oxo-4-phthalimido-5-isoxazolidine acetic acid,m.p. 160°-2° (ethyl acetate/hexane, 1:1).

A mixture of the two acids is found in Fraction 15.

Using the same procedure as in Preparation 26 but substituting(2R,3S)-t-butyl-[2-phthalimido-3-cyclopenten-1yl)oxy]carbamate for(2R,3S)-benzyl-[2-phthalimido-3-cyclopenten-1-yl)oxy]carbamate there isobtained(α5,5S)-2-[(tbutyloxy)carbonyl]-3-oxo-α-phthalmido-5-isoxazolidineacetic acid and(4S,5S)-2-[(t-butyloxy)-carbonyl]-3-oxo-4-phthalimido-5-isoxazolidineacetic acid.

NMR (CDCl₃ ,δ): 1.35 (s,9), 2.7-3.0 (m, 2), 4.55-5.0 (m, 1), 5.15-5.55(m, 1), 5.55-6.25 (m, 2), 7.2-7.4(1), 7.6-8.0 (m, 4).

Preparation 27 (α5,5S)-3-oxo-α-phthalimido-5-isoxazolidine acetic acid##STR71##

A 7.48 g. (17.6 mM) quantity of (αS,5S)2-[(benzyloxy)carbonyl]-3-oxo-α-phthalimido-5-isoxazolidine acetic acidis dissolved in 150 ml. of ethyl acetate and 75 ml. of 95% ethanol. Thesolution is treated with 1.5 g. of palladium black and hydrogenated at25° C. and 1 atmosphere of pressure. The reaction is stopped after 165minutes, filtered, and the filtrate is evaporated in vacuo to yield3-oxo-α-phthalimido-5-isoxazolidine acetic acid as a residue.

NMR (CD₃ OD, δ): 2.9 2(partially split d, CH₂), 5.1-5.7 (m, 2), 7.85 (s,4 ArH).

TLC (silica gel 60): Rf=0.36 in the upper phase of (9-2-5-10)ethylacetate-acetic acid cyclohexane water. In the same system thestarting material Rf=0.46 and the hydrogenated product from 33 Rf=0.54,and compound 33 Rf=0.64.

Preparation 28 (α5,5S)-3-Oxo-α-phthalimido-5-isoxazolidine acetic acid,benzhydryl ester 15b ##STR72##

A 2.8 g (14.3 mM) quantity of benzophenone hydrazone was dissolved in 80ml of ethyl ether. The solution is treated with 10 g of sodium sulfate,6.02 g (28 mM) of yellow mercury oxide and 0.2 ml of a saturatedpotassium hydroxide solution in ethyl alcohol. The reaction is stirred60 minutes and the resultant deep burgandy mixture filtered. Theresultant solution is added directly to the reaction solution from a8.45 mM hydrogenation in which(α5,5S)-3-oxo-α-phthalimide-5-isoxazolidine acetic acid is prepared.After one hour the reaction is treated with sufficient 3 N hydrochloricacid while stirring vigorously, to destroy the excess burgandy coloreddiazo compounds. The reaction mixture is then evaporated in vacuo (<30mm and <30° C.), and chromatographed over 300 g. of CC-4 silica geleluted with (40-60) ethyl acetate-toluene. Thirty five ml. fractions arecollected. The (αS,5S) 3-oxo-α-phthalimido-5-isoxazolidineacetic acid,benzhydryl ester is found in fr actions 10-14 [α]_(D) =+26° (C=0.70).

NMR (CDCl₃ δ): 2.75 (d, J=11.5, CH₂), 5.15-5.7 (m, 2), 6.93 (s, CH (C₆H₅)₂), 7.18 (s, C₆ H₅), 7.28 (s, C₆ H₅), 7.5-8.0 (m, 4, ArH).

TLC (silica gel 60): Rf=0.42 in (40-60) ethyl acetatetoluene. Theisoproduct from compound VIIb has Rf=0.59 in this same system.

Preparation 29 Tricholomic Acid ##STR73##

To 1.20 g. (6.75 mmole) of(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazol-acetic acid (AT-125), isadded 20 ml. of 2 N sodium hydroxide and stirred at 25° for 36 hours.The solution is brought to ˜3.5 pH with 6 N hydrochloric acid (dropwiseaddition) and then stored at -10° (freezer) overnight. The crystals arecollected by filtration, washed twice with cold water, and then driedgiving 720 mg. of trichloromic acid. The initial filtrate is lyophilizedand the resulting powder is taken up in 4 ml. of water, filtered, andwashed with two 4 ml. portions of cold water, and dried yielding anextra 160 mg. of tricholomic acid (total 80% yield).

NMR (D₂ O): 5.65-5.35 (1H, m, ring CH), 4.27 (1H, d, J=3.5 H₃, α-H),3.69 (2H, d, J=9.5 H3, CH₂).

TLC: Rf=0.31 (ninhydrin), (solvent: 60% methylethyl ketone, 20% acetone,15% water, 5% acetic acid).

C-NMR (D₂ O): 154.6 (CONH), 79.4 (CHO), 56.8 (α-C), 36.8 (CH₂).

Preparation 30 Phthalyl-tricholomic acid, methyl ester ##STR74##

A 400 mg. (2.5 mmole) sample of tricholomic acid, 720 mg. of sodiumcarbonate, 6 ml. water, and 1.2 g. of N-carbethoxyphthalimide are mixedwith the latter added in 3 portions over first 2 hours. Afteracidification to pH 3 with 3 N HCl the reaction mixture is washed with2×25 ml. of ethyl acetate. The combined organic phases are washed withbrine, dried over sodium sulfate and concentrated in vacuo.Chromatography (CC-4, 50→75% E/H gradient) yields phthalyl-tricholomicacid.

NMR (methanol d₄): 7.86 (4H, s, φ), 5.65-5.0 (2H, m, CHO, α-H), 2.92(2H, d, CH₂).

TLC: (AIX) Rf=0.15 (UV; yellow stain with vanillin/H₃ PO₄ spray,Δ).

The product is taken up to 10 ml. of dry tetrahydrofuran and treatedwith etheral diazomethane. Chromatography (silica gel 75% E/H afforded160 mg.

NMR (CDCl₃): 5.65-5.3 (1H, m, CHO), 5.3-5.1 (1H, m, α-H), 3.06 (2H, d,CH₂, PHTH unchanged.

TLC: (AIX)Rf=0.33 (50% E/H) Rf-0.15; (AT-125 derivative) Rf=0.42.

Analysis: Calc. C, 55.26; H, 3.98; N, 9.21. Found: C, 55.20; H, 4.60; N,8.43.

Mass Spectrometry: M⁺ m/e 304 (3%), 272 (H⁺ -CH₂ OH), 244 (M⁺ -CO₂ CH₃),219.

Example 1 (αS,5S)-Chloro-4,5-dihydro-α-phthalimido-5-isoxazole aceticacid, benzhydryl ester ##STR75##

To 192 μl (2 mmole) of carbon tetrachloride in 25 ml of drytetrahydrofuran under N₂ at room temperature is added 310 μl (1.9 mmole)of hexamethylphosphorous triamide dropwise over one minute. Within twominutes 590 mg. (1.3 mmole) of (αS,5S)-oxo-α-phthalimido-5-isoxazolidineacetic acid, benzhydryl ester acid in approximately 5 ml of dry THF isadded rapidly. The heterogeneous solution is stirred at 45° for 48hours. Alternatively, to 590 mg. (1.3 mmole) of(αS,5S)-oxo-α-phthalimido-5-isoxolidine in 20 ml. of dry tetrahydrofuranunder N₂ at room temperature is added 610 mg. (2.6 mmole) ofhexamethylphosphorous triamide dichloride (prepared by hexachloroethanein acetonitrile with hexamethylphosphorous triamide according to R.Appel and H. Scholer, Chem. Ber., 110, 2382 (1977)). The solution isrefluxed for 48 hours. The reaction is taken up in 150 ml of ethylacetate and washed with 50 ml of 0.1 N hydrochloric acid followed bybrine and then dried over sodium sulfate. After concentrating in vacuothe residue is chromatographed on 50 g of silica gel with 30% ethylacetate hexane (200 ml elution) which yields 200 mg of crude product(33% yield), 245 mg of starting material (42%). The crude productyielded (α5,5S) 3-chloro-4,5-dihydro-α-phthalimido-5-isoxazole aceticacid benzhydryl ester as needles, m.p. 178°-9° upon recrystallizationfrom methanol.

Tlc: Rf (product)=0.70 in 35% ethyl acetate/hexane (S.M.)=0.11

(α)₂₀ (CHCl₃)+79° (589 nm); 82° (578); 94° (547); 163° (436); 256°(365).

NMR (CDCl₃): 7.80 (4H, m, PHTH), 7.32 and 7.22 (10H, s, φ), 6.95 (1H, s,φCH), 5.6 (1H, m, CHO), 5.26 (1H, d, α-H), 3.32 (2H, d, CH₂).

Analysis: Calc'd. for C₂₆ H₁₉ ClN₂ O₅ : C, 65.75; H, 4.03; N, 5.90.Found: C, 65.45; H, 4.08; N, 5.95.

Using the procedures described above but substituting the(αS,5S)-3-oxo-4,5-dihydro-4β-phthalimido-5α-isoxazole acetic acid,benzhydryl ester (VIIb) affords the (αS,5S) (resp.)3-chloro-4,5-dihydro-4β-phthalimido-5α-isoxazole acetic acid, benzhydrylester (VIIIc, X=Cl).

Example 2 (αS,5S)-Chloro-4,5-dihydro-α-phthalimido-5-isoxazole aceticacid ##STR76##

To 185 mg (0.4 mmole) of 3-chloro-4,5-dihydro-α-phthalimido-5-isoxazoleacetic acid, benzhydryl ester in 3 ml of dry nitromethane in an ice bathunder nitrogen is bubbled dry hydrogen chloride five minutes. The bathis removed and the solution stirred for one hour. After concentratingthe solution in vacuo the residue is chromatographed on CC-4 silica gel(30 g) with 30% ethyl acetate/hexane to yield 115 mg of a white foam.This foam is recrystallized from 4/1 hexane/ethanol to yield(αS,5S)-3-chloro-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid, am.p. 176°-7° C. (forms a hydrate on exposure to moisture).

Tlc: Rf=0.45 (AIX)

NMR: (Acet-d₆) 7.95 (4H, s, PHTH), 5.6 (1H, m, CHO), 5.27 (1H, d, α-H),3.57 (2H, d, CH₂).

Analysis: Calc'd for C₁₃ H₉ ClN₂ O₅ : C, 50.58; H, 2.94; N, 9.08. Found:C, 50.57; H, 3.06; N, 9.46.

Using the same procedure as described above but substituting(αS,5S)-3-chloro-4,5-dihydro-4β-phthalimido-5α-isoxazole acetic acid,benzhydryl ester (VIIIc, X=Cl) affords (αS,5S) (resp.)3-chloro-4,5-dihydro-4β-phthalimido-5α-isoxazole acetic acid (VIIIb,X×Cl).

Example 3 (αS,5S)-Bromo-4,5-dihydro-α-phthalimido-5-isoxazole aceticacid ##STR77##

To 185 mg (0.4 mmole) of the(αS,5S)-chloro-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid,benzhydryl ester in 3 ml of dry nitromethane in an ice bath undernitrogen is bubbled dry hydrogen bromide for five minutes. The bath isremoved and the solution stirred for one hour. After concentrating thesolution in vacuo the residue is chromatographed on CC-4 silica gel (30g) with 30%) ethyl concentrating the solution in vacuo the residue ischromatographed on CC-4 silica gel (30 g) with 30%) ethyl acetate/hexaneto yield 115 mg of a white foam. This foam is recrystallized from 4/1hexane/ethanol to yield 3-bromo-4,5-dihydro-α-phthalimido-5-isoxazoleacetic acid.

Analysis: Calc'd. for C₁₃ H₉ BrN₂ O₅ : C, 44.21; H, 2.57; N, 7.93.Found: C, 44.49; H, 2.64; N, 7.83. [α]₂₀ (MeOH) 67° (589 nm); 70° (578);79° (547); 131° (436); 187° (365).

Utilizing the same procedure as in Example 3 but substituting hydrogeniodide for hydrogen bromide there is obtained3-iodo-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid.

Again utilizing the same procedure as in Example 3 but substitutinghydrogen floride for hydrogen bromide there is obtained3-fluoro-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid.

Example 4phthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acidmethyl ester from phthalyltricholomic acid methyl ester ##STR78##

To 60 mg. (0.2 mmole) of tricholomic acid ester phthalimide in 1 ml. ofdry tetrahydrofuran at room temperature under N₂ is added 25 μl ofcarbon tetrachloride followed by 30 μl of hexamethylphosphoroustriamide. After 16 hours another 10 μl of carbon tetrachloride and 10 μlof phosphine were added. The solution was worked up after a totalreaction time of 36 hours with ethyl acetate/water. The products arechromatographed on 5 g. silica gel (50% E/H) to recover 35 mg. ofphthalyl-(α5,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazol-acetic acidmethyl ester.

NMR: (CDCl₃) identical to AT-125-PHTH methyl ester, prepared fromnatural (+) AT-125 followed by Preparation 30.

TLC: identical to AT-125-PHTH methyl ester (UV and staining identical).

Example 5 (αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acidor AT-125 ##STR79##

To 710 mg (2.3 mmole) of 3-chloro-4,5-dihydro-α-phthalimido-5-isoxazoleacetic acid in 15 ml of water is added 260 μl of hydrazine hydrate andthe solution stirred at 50° for 7 hours. After cooling, the solution isadjusted to pH 5.5 with acetic acid (80 μl), filtered, and theprecipitate washed with 13 ml water. The filtrate is diluted with 200 mlof 2-butanol and allowed to crystallized in the refrigerator for 18hours. Filtration yielded 240 mg of(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid;concentrating the mother liquors approximately 15% afforded 50 mg of asecond crop of (αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole aceticacid (71%).

Tlc: Rf 0.40 (60/20/15/5; MEK/Acet/H₂ O/AcOH).

Nmr: (D₂ O) 5.2 (1H, m, CHO), 4.17 (1H, d, α-H), 3.61 (2H, d, CH₂).

Using the same procedure as described above but substituting DL- orD-3-chloro-4,5-dihydro-4β-phthalimido-5α-isoxazole acetic acid (VIIIb),X=Cl) affords DL- orD-(resp.)-3-chloro-4,5-dihydro-4β-amino-5α-isoxazole acetic acid(VIIIa). A similar substitution of IIb for Ib affords tricholomic acid.

Analysis: Calc'd. for C₅ H₉ ClN₂ O₃ : C, 33.63; H, 3.95; N, 15.69; Cl,19.86. CD: θ₂₁₆ ^(max) =13,300

Found: C, 33.60; H, 4.11; N, 16.15; Cl, 19.63.

Example 6 (αS,5S)-α-amino-3-bromo-4,5-dihydro-5-isoxazole acetic acid##STR80##

To 710 mg (2.3 mmole) of 3-bromo-4,5-dihydro-α-phthalimido-5-isoxazoleacetic acid in 15 ml of water is added 260 μl of hydrazine hydrate andthe solution stirred at 50° for 7 hours. After cooling, the solution isadjusted to pH 5.5 with acetic acid (80 μl), filtered, and theprecipitate washed with 13 ml water. The filtrate is diluted with 200 mlof 2-butanol and allowed to crystallized in the refrigerator for 18hours. Filtration yielded 240 mg of(αS,5S)-α-amino-3-bromo-4,5-dihydro-5-isoxazole acetic acid;concentrating the mother liquors approximately 15% afforded 50 mg of asecond crop of (αS,5S)-α-amino-3-bromo-4,5-dihydro-5-isoxazole aceticacid (71%).

The tlc and nmr were indistinguishabel from the chloro-analog. The UVspectrum exhibited the same extinction but at 214 nm.

CD: θ₂₁₃ ^(max) =11,250

Analysis: Calc'd for C₅ H₇ BrN₂ O₃ : C, 26.92; H, 3.16; N, 12.56; Br,15.73. Found: C, 26.63; H, 3.33; N, 12.60; Br, 15.58. (Br determinedfrom Cl coulometric assay)

MS: on disilyl compound indicates M⁺ -15 at 352 and 354 m/e.

Example 7 Direct conversion of Tricholomic Acid to AT-125 ##STR81##

To 140 mg. of tricholomic acid in 3 ml. POCl₃ under N₂ at roomtemperature was added 160 μl of purified diethylaniline. The mixture wasplaced in a 100° bath for 5 minutes. The POCl₃ was removed in vacuo. Theresultant solid was treated with 2 ml. H₂ O and stirred for 10 minuteswhile neutralizing with concentrated NH₄ OH. After lyophilization thematerial was applied to a 10 g. silica gel column and eluted with 85%EtOAc/5% H₂ O/10% acetic acid. A dark band with TLC mobility similar toAT-125 was recovered. The material was bio-assayed against B. subtilis(syn.) and found to exhibit a minimum of 2% yield (assuming thatstarting tricholomic acid is 100% pure and knowing the bioactivity oftricholomic acid).

Example 8 (αS,5S)-3-methoxy-4,5-dihydro-α-phthalimido-5-isoxazole aceticacid, benzhydryl ester ##STR82##

To 90 mg. (0.2 mmole) of 3-oxo-α-phthalimido 5-isoxazolidine aceticacid, benzhydryl ester (IIa) in 5 ml of methylene chloride is added a50% molar excess of a standard etheral diazomethane solution. A 5 μl.amount of βF₃.Et₂ O is added and, after 30 minutes, the reaction isconcentrated and the residue chromatographed on preparative TLC (AIXeluent) to yield (45 mg.) of3-methoxy-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid, benzhydrylester.

NMR (CDCl₃): 7.80 (m, 4H), 7.32 and 7.22 (s, 10H, Ph), 6.96 (s, Ph₂ CH),5.1-5.8 (m, 2H), 3.75 (s, CH₃ O), 3.07 (d, J=7.5 Hz, 2H)

TLC (AIX)=Rf=0.60

A second product is produced (35 mg) having the structure ##STR83##

NMR (CDCl₃): 7.82 (m, 4H), 7.32 and 7.22 (s, 10H), 6.97 (s, 1H), 5.1-5.9(m, 2H), 3.10 (s, NCH₃), 2.81 (m, 2H)

TLC A IX): Rf=0.40

Example 9Phthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid,methyl ester ##STR84##

To 110 mg. (0.35 mM) ofphthalyl-(α-S,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazol acetic acid in3 ml. of ether is added ethereal diazomethane until gas evolution ceasedand the yellow color persists. Concentration and chromatography on 15 gof silica gel (40% ethylacetate/hexane to yields 110 mg ofphthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid,methyl ester as an oil (95%).

NMR (CDCl₃): 7.87 (4H, m, φ), 5.6 (1H, m, CHO), 5.22 (1H, d, J=6 Hz,α-H), 3.78 (3H, s, CH₃), 3.40 (2H, d, J=9 Hz, CH₂).

TLC: (AIX) Rf=0.50, (85% E/H) Rf=0.70.

¹³ C NMR: (CDCl₃) 167.5 (CO₂), 167.1 (CON), 149.9 (C-Cl), 134.7, 131.5,124.0 (φ), 80.2 (CHO), 53.1 (CH₃), 52.9 (α-C), 41.5 (CH₂).

Analysis: Calc'd C, 52.10; H, 3.44; N, 8.68. Found: C, 52.58; H, 3.67;N, 8.39.

Mass spectrometry: no M⁺ m/e 322, 291 (M⁺ -OCH₃), 287 (M⁺ -Cl), 263 (M⁺-CO₂ CH₃), 219 (62%, PHTH-COCHO₂ CH₃).

Example 103-Chloro-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-4,5-dihydro-5-isoxazoleacetic acid

To 178 mg. of AT-125 in 4 ml. of water is added 300 mg. of sodiumbicarbonate. After cooling the mixture to 0°-5° C., 300 mg. of9-hydroxymethyl fluorenylcarbonyl chloride is added and it is stirredfor two hours and allowed to stand overnight at room temperature. Theresulting semi solid is taken up in ethyl acetate and 1 N hydrochloricacid is added until a pH of 3 is attained. The organic phase isseparated, dried over sodium sulfate, concentrated and chromatographedon 20 g. of CC-4 silica gel employing 40% ethyl acetate/Hexane. Thosefractions containing the desired product are evaporated and the residuerecrystallized from ethyl acetate to yield3-chloro-α-[[(9H-fluoren-9-yl-methoxy)carbonyl]amino]-4,5-dihydro-5-isoxazoleacetic acid, m.p. 182°-183° C. The NMR fits the desired structure.

Example 11 Phthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, p-methoxybenzyl ester ##STR85##

To 33 mg (0.1 mmole) ofphthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid in2 ml of dry acetonitrile is added 50 μl of diisopropylethylaminefollowed by 30 mg of 4-methoxybenzyl bromide. After stirring at roomtemperature under nitrogen for 6 hours, the reaction solution is takenup in ethyl acetate and 0.5 hydrochloric acid. The organic phase isseparated, dried over sodium sulfate, and concentrated in vacuo. Theresulting residue is chromatographed on preparative-layer silica gelplates with 40% ethyl acetate/hexane eluent. The desired band isremoved, yielding 35 mg (85%) ofphthalyl-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid,p-methoxybenzyl ester.

NMR (CDCl₃): 7.84 (4H, m, PHTH), 7.26 and 6.83 (4H, A₂ B₂, φ), 5.6 (1H,m, CHO), 5.2 (1H, d, J=6 Hz, α-H), 4.64 (2H, s), 3.78 (3H, s), 3.38 (2H,d, J=9 Hz).

We claim:
 1. A process for preparing racemic mixtures and opticallyactive isomers of a compound having the formula ##STR86## whichcomprises chlorinating a compound having the formula ##STR87## wherein Ris selected from the group consisting of hydrogen, alkyl of from 1 to 8carbon atoms, inclusive, halogenated alkyl of from 1 to 3 halogen atoms,and 1 to 5 carbon atoms, inclusive and aralkyl of from 7 to 20 carbonatoms, inclusive and substituted aralkyl of from 7 to 20 carbon atoms,inclusive; and R₁₄ and R₁₅ are selected from the group consisting ofhydrogen, ##STR88## inclusive, or when taken together with the nitrogenatom form the group ##STR89## wherein R₆ is alkyl of from 1 to 8 carbonatoms, inclusive, halogenated alkyl of from 1 to 5 carbon atoms,inclusive, and 1 to 3 halogen atoms, inclusive, aralkyl of from 7 to 20carbon atoms, inclusive, and substituted aralkyl of from 7 to 20 carbonatoms, inclusive, R₇ is selected from the group consisting of alkyl offrom 1 to 12 carbon atoms, inclusive, aryl of from 6 to 20 carbon atoms,inclusive, aryl of from 6 to 20 carbon atoms, inclusive, aralkyl of from7 to 20 carbon atoms, inclusive, substituted aralkyl of from 7 to 20carbon atoms, inclusive, and R₈ is selected from the group consisting of##STR90## where R₉, R₁₀, R₁₁ and R₁₂ are selected from the groupconsisting of hydrogen and alkyl of from 1 to 5 carbon atoms, inclusive,(b) ##STR91## (c) orthointerphenylene, and (d) substitutedorthointerphenylene.
 2. A process according to claim 1 wherein thecompound prepared has the formula ##STR92## and the compound chlorinatedhas the formula ##STR93## wherein R and R₈ are the same as in claim 1.3. A process according to claim 2 wherein R₈ is orthointerphenylene. 4.A process according to claim 3 wherein R is benzhydryl so that thecompound prepared is(αS,5S)-chloro-4,5-dihydro-α-phthalimido-5-isoxazole acetic acid,benzhydryl ester.
 5. A process according to claim 1 wherein the compoundprepared has the formula ##STR94## and the compound chlorinated has theformula ##STR95## wherein R is the same as in claim
 1. 6. A processaccording to claim 5 wherein R is hydrogen so that the compound preparedis (αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid.
 7. Aprocess according to claim 3 wherein R is methyl and R₈ isorthointerphenylene, so that the compound prepared is phthalyl(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid methylester.
 8. A process according to claim 2 or 7 wherein the chlorinatingagent comprises carbon tetrachloride and hexamethylphosphorous in thepresence of a solvent.
 9. A process according to claim 2 or 7 whereinthe chlorinating agent comprises hexamethylphosphorous triamidedichloride.
 10. A process according to claim 5 or 6 wherein thechlorinating agent comprises phosphorous oxychloride.
 11. A processaccording to claim 1 wherein the compound prepared is optionally treatedwith hydrogen bromide to yield a compound having the formula ##STR96##12. A process according to claim 4 wherein the compound prepared isoptionally treated with hydrogen bromide to yield(αS,5S)-bromo-4,5-dihydro-α-phthalimido-5-isoxazole acetic acidbenzhydryl ester.